Xanthine dehydrogenase (xdh) irna compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting a xanthine dehydrogenase (XDH) gene, and methods of using such double stranded RNAi agents to inhibit expression of an XDH gene and methods of treating subjects having an XDH-associated disease.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/752,742, filed on Jan. 27, 2020, which is a continuation of U.S.patent application Ser. No. 15/747,571, filed on Jan. 25, 2018,abandoned, which is a 35 U.S.C. § 371 national stage filing ofInternational Application No. PCT/US2016/043985, filed on Jul. 26, 2016,which in turn claims the benefit of priority to U.S. ProvisionalApplication No. 62/287,522, filed on Jan. 27, 2016, U.S. ProvisionalApplication No. 62/255,603, filed on Nov. 16, 2015, and U.S. ProvisionalApplication No. 62/197,221, filed on Jul. 27, 2015. The entire contentsof each of the foregoing applications are hereby incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Feb. 1, 2022, isnamed 121301_04006_SL.txt and is 600,597 bytes in size.

BACKGROUND OF THE INVENTION

Reduced renal clearance of uric acid is the result of a number offactors, including defects in uric acid transporter proteins such asSLC2A9, ABCG2, and others; reduced renal excretion due to renal disease,hypothyroidism, volume contraction and volume depletion, acidosis, leadintoxication, and familial nephropathy due to uromodulin deposits; andaltered renal clearance due to hyperinsulinemia or insulin resistance indiabetes. Increased synthesis of uric acid is associated withhyperuricemia plus hyperuricosuria; inborn errors of metabolism such asLesch Nyhan/HPRT deficiency, PRPP synthetase overactivity, andglucose-6-phosphate dehydrogenase deficiency (Von Gierkedisease/Glycogen Storage Disease Type Ia); certain situations of highcell turnover (e.g., tumor lysis syndrome); certain situations of highATP turnover (e.g., glycogen storage diseases, tissue ischemia).Furthermore, conditions such as chronic kidney disease, hypertension,metabolic syndrome, and high fructose intake may result in bothincreased uric acid synthesis and decreased uric acid clearance.

Chronic elevated serum uric acid (chronic hyperuricemia), typicallydefined as serum urate levels greater than 6.8 mg/dl (greater than 360mmol/), the level above which the physiological saturation threshold isexceeded (Mandell, Cleve. Clin. Med. 75:S5-S8, 2008), is associated witha number of diseases. For example, gout is characterized by recurrentattacks of acute inflammatory arthritis that is caused by aninflammatory reaction to uric acid crystals in the joint typically dueto insufficient renal clearance of uric acid or excessive uric acidproduction. Fructose associated gout is associated with variants oftransporters expressed in the kidney, intestine, and liver. Chronicelevated uric acid is also associated with non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), metabolic disorder,cardiovascular disease, type 2 diabetes, and conditions linked tooxidative stress, chronic low grade inflammation, and insulin resistance(Xu et al., J. Hepatol. 62:1412-1419, 2015; Cardoso et al., J. Pediatr.89:412-418, 2013; Sertoglu et al., Clin. Biochem., 47:383-388, 2014).

Uric acid (also referred to herein as urate) is the final metabolite ofendogenous and dietary purine metabolism. Xanthine oxidase (XO) (EC1.1.3.22) and xanthine dehydrogenase (XDH) (EC 1.17.1.4), which catalyzethe oxidation of hypoxanthine to xanthine, and xanthine to uric acid,respectively, are interconvertible forms of the same enzyme. The enzymesare molybdopterin-containing flavoproteins that consist of two identicalsubunits of approximately 145 kDa. The enzyme from mammalian sources,including man, is synthesized as the dehydrogenase form, but it can bereadily converted to the oxidase form by oxidation of sulfhydrylresidues or by proteolysis. XDH is primarily expressed in the intestineand the liver, but it is also expressed in other tissues includingadipose tissue.

Allopurinol and febuxostat (Uloric®), inhibitors of the XDH form of theenzyme, are commonly used for the treatment of gout. However, their useis contraindicated in patients with co-morbidities common to gout,especially decreased renal function, e.g., due to chronic kidney diseaseor hepatic impairment. Their use may also be limited in patients withmetabolic syndrome, hypertension, dyslipidemia, non-alcoholicsteatohepatitis (NASH) or non-alcholic fatty liver disease (NAFLD),cardiovascular disease, or diabetes (either type 1 or type 2), due tolimited organ function from the disease or condition, or due to adversedrug interactions with agents used for the treatment of such conditions.

Currently, treatments for gout do not fully meet patient needs.Therefore, there is a need for additional therapies for subjects thatwould benefit from reduction in the expression of an XDH gene, such as asubject having an XDH-associated disease or disorder, e.g., gout.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an XDH gene. The XDH gene may be within a cell, e.g., acell within a subject, such as a human.

In an aspect, the invention provides a double stranded ribonucleic acid(dsRNA) agent for inhibiting expression of a xanthine dehydrogenase(XDH) gene, wherein the dsRNA agent comprises a sense strand and anantisense strand, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1 or 9 and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2 or 10,respectively.

In certain embodiments, the sense strand and antisense strandindependently comprise sequences selected from the group consisting ofany one of the sequences in any one of Tables 3, 4, 6, and 7.

In an aspect, the invention provides a double stranded ribonucleic acid(dsRNA) agent for inhibiting expression of xanthine dehydrogenase (XDH),wherein the dsRNA agent comprises a sense strand and an antisensestrand, the antisense strand comprising a region of complementaritywhich comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the antisense sequences listed in anyone of Tables 3, 4, 6, and 7.

In certain embodiments, the dsRNA agent comprises at least onenucleotide comprising a nucleotide modification. In one embodiment,substantially all of the nucleotides of the sense strand comprise amodification. In another embodiment, substantially all of thenucleotides of the antisense strand comprise a modification. In yetanother embodiment, substantially all of the nucleotides of the sensestrand and substantially all of the nucleotides of the antisense strandcomprise a modification. In one embodiment, all of the nucleotides ofthe sense strand comprise a modification. In another embodiment, all ofthe nucleotides of the antisense strand comprise a modification. In someembodiments, all of the nucleotides of the sense strand and all of thenucleotides of the antisense strand comprise a nucleotide modification.

In an aspect, the invention provides a double stranded ribonucleic acid(dsRNA) agent for inhibiting expression of a xanthine dehydrogenase(XDH) gene, wherein the dsRNA agent comprises a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 or 9 andthe antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:2 or 10, respectively, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand comprise a nucleotide modification, and whereinthe sense strand is conjugated to a ligand attached at the 3′-terminus.

In certain embodiments, the present invention provides double strandedribonucleic acid (dsRNA) agents for inhibiting expression of a XDH gene,which comprise a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of nucleotides 490-512, 86-104, 273-291, 339-358, 410-428, 444-462,490-509, 493-512, 888-907, 936-954, 969-987, 1105-1123, 1158-1176,1242-1260, 1326-1344, 1357-1412, 1357-1379, 1357-1376, 1360-1379,1378-1412, 1378-1396, 1394-1412, 1481-1499, 1515-1548, 1515-1533,1530-1548, 1718-1736, 1783-1802, 1854-1890, 1854-1872, 1872-1890,2053-2072, 2077-2096, 2137-2160, 2137-2156, 2142-2160, 2173-2206,2173-2192, 2177-2195, 2184-2203, 2187-2206, 2314-2332, 2567-2604,2567-2585, 2585-2604, 2620-2640, 2620-2639, 2621-2640, 2722-2740,2891-2909, 2941-2975, 2941-2959, 2956-2975, 2993-3011, 3025-3061,3025-3044, 3042-3061, 3062-3110, 3062-3080, 3079-3097, 3091-3110,3112-3147, 3112-3130, 3129-3147, 3197-3215, 3247-3265, 3316-3334,3366-3384, 3487-3520, 3487-3505, 3502-3520, 3606-3624, 3672-3690,3891-3930, 3891-3910, 3893-3912, 3912-3930, 4063-4081, 4114-4132,4152-4171, 4200-4218, 4300-4337, 4300-4319, 4303-4321, 4319-4337,4386-4404, 4519-4538, 4541-4559, 4618-4637, or 4703-4722 of thenucleotide sequence of SEQ ID NO:1.

In certain embodiments, the present invention provides double strandedribonucleic acid (dsRNA) agents for inhibiting expression of a XDH gene,which comprise a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 15contiguous nucleotides of any one of nucleotides 490-512, 86-104,273-291, 339-358, 410-428, 444-462, 490-509, 493-512, 888-907, 936-954,969-987, 1105-1123, 1158-1176, 1242-1260, 1326-1344, 1357-1412,1357-1379, 1357-1376, 1360-1379, 1378-1412, 1378-1396, 1394-1412,1481-1499, 1515-1548, 1515-1533, 1530-1548, 1718-1736, 1783-1802,1854-1890, 1854-1872, 1872-1890, 2053-2072, 2077-2096, 2137-2160,2137-2156, 2142-2160, 2173-2206, 2173-2192, 2177-2195, 2184-2203,2187-2206, 2314-2332, 2567-2604, 2567-2585, 2585-2604, 2620-2640,2620-2639, 2621-2640, 2722-2740, 2891-2909, 2941-2975, 2941-2959,2956-2975, 2993-3011, 3025-3061, 3025-3044, 3042-3061, 3062-3110,3062-3080, 3079-3097, 3091-3110, 3112-3147, 3112-3130, 3129-3147,3197-3215, 3247-3265, 3316-3334, 3366-3384, 3487-3520, 3487-3505,3502-3520, 3606-3624, 3672-3690, 3891-3930, 3891-3910, 3893-3912,3912-3930, 4063-4081, 4114-4132, 4152-4171, 4200-4218, 4300-4337,4300-4319, 4303-4321, 4319-4337, 4386-4404, 4519-4538, 4541-4559,4618-4637, or 4703-4722 of the nucleotide sequence of SEQ ID NO: 1.

In certain embodiments, the sense strand and the antisense strandcomprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense sequences listed in any one of Tables 3, 4, 6, and7. For example, in certain embodiments, the sense strand and theantisense strand comprise a region of complementarity which comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of the antisense sequences of any one of the duplexes inany one of Tables 3, 4, 6 and 7. In certain embodiments, the sensestrand and the antisense strand comprise a region of complementaritywhich comprises at least 15 contiguous nucleotides of any one of theantisense sequences of the duplexes any one of the duplexes in any oneof Tables 3, 4, 6 and 7.

For example, in certain embodiments, the sense strand and the antisensestrand comprise a region of complementarity which comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense nucleotide sequences selected from the groupconsisting of the antisense nucleotide sequence of any of of duplexesAD-70016, AD-70033, AD-70053, AD-70050, AD-70051, AD-72007, AD-71990,AD-70049, AD-71993, AD-70042, AD-70052, AD-70018, AD-70055, AD-71831,AD-71810, AD-70023, AD-70035, AD-70044, AD-71820, AD-70027, AD-70034,AD-71837, AD-71861, AD-71998, AD-70020, AD-70026, AD-70030, AD-71801,AD-72003, AD-70025, AD-70021, AD-70029, AD-71834, AD-71765, AD-70043,AD-71840, AD-72006, AD-71844, AD-71779, AD-71830, AD-71952, AD-71766,AD-71950, AD-70022, AD-71833, AD-71823, AD-71847, AD-71900, AD-72014,AD-72015, AD-70037, AD-71982, AD-71787, AD-71894, AD-70048, AD-71887,AD-70028, AD-71980, AD-71826, AD-71855, AD-71778, AD-71757, AD-72012,AD-71854, AD-71890, AD-70038, AD-71865, AD-71933, AD-71942, AD-71901,AD-71878, AD-71905, and AD-71914.

In certain embodiments, the sense and antisense nucleotide sequences ofa dsRNA agent of the invention are selected from the group consisting ofa duplex selected from the group consisting of duplexes AD-70016,AD-70033, AD-70053, AD-70050, AD-70051, AD-72007, AD-71990, AD-70049,AD-71993, AD-70042, AD-70052, AD-70018, AD-70055, AD-71831, AD-71810,AD-70023, AD-70035, AD-70044, AD-71820, AD-70027, AD-70034, AD-71837,AD-71861, AD-71998, AD-70020, AD-70026, AD-70030, AD-71801, AD-72003,AD-70025, AD-70021, AD-70029, AD-71834, AD-71765, AD-70043, AD-71840,AD-72006, AD-71844, AD-71779, AD-71830, AD-71952, AD-71766, AD-71950,AD-70022, AD-71833, AD-71823, AD-71847, AD-71900, AD-72014, AD-72015,AD-70037, AD-71982, AD-71787, AD-71894, AD-70048, AD-71887, AD-70028,AD-71980, AD-71826, AD-71855, AD-71778, AD-71757, AD-72012, AD-71854,AD-71890, AD-70038, AD-71865, AD-71933, AD-71942, AD-71901, AD-71878,AD-71905, and AD-71914. In certain embodiments, the sense strand and theantisense strand comprise a region of complementarity which comprises atleast 15 contiguous nucleotides of any one of the sense and antisensenucleotide sequences of any one of the foregoing duplexes.

In some embodiments, all of the nucleotides of the sense strand and allof the nucleotides of the antisense strand comprise a nucleotidemodification. In one embodiment, the modified nucleotides areindependently selected from the group consisting of a deoxy-nucleotide,a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modifiednucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an unlocked nucleotide, aconformationally restricted nucleotide, a constrained ethyl nucleotide,an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic. In another embodiment, the modifiednucleotides comprise a short sequence of 3′-terminal deoxy-thyminenucleotides (dT), e.g., 1, 2, or 3 3′-terminal deoxy-thymine nucleotides(dT). In one embodiment, the modified nucleotides comprise two3′-terminal deoxy-thymine nucleotides (dT)

In certain embodiments, substantially all of the nucleotides of thesense strand are modified. In certain embodiments, substantially all ofthe nucleotides of the antisense strand are modified. In certainembodiments, substantially all of the nucleotides of both the sensestrand and the antisense strand are modified.

In certain embodiments, the duplex comprises, or consists of, a modifiedantisense strand nucleotide sequence provided in any one of Tables 4 and7. In certain embodiments, the duplex comprises a modified sense strandnucleotide sequence provided in Table 4. In certain embodiments, theduplex comprises any one of the modified sense strand and antisensestrand nucleotide sequences of a duplex provided in any one of Tables 4and 7.

In certain embodiments, the region of complementarity between theantisense strand and the target is at least 17 nucleotides in length.For example, the region of complementarity between the antisense strandand the target is 19 to 21 nucleotides in length, for example, theregion of complementarity is 21 nucleotides in length. In preferredembodiments, each strand is no more than 30 nucleotides in length.

In some embodiments, at least one strand comprises a 3′ overhang of atleast 1 nucleotide, e.g., at least one strand comprises a 3′ overhang ofat least 2 nucleotides.

In many embodiments, the dsRNA agent further comprises a ligand. Theligand can be conjugated to the 3′ end of the sense strand of the dsRNAagent. The ligand can be an N-acetylgalactosamine (GalNAc) derivativeincluding, but not limited to

In various embodiments, the ligand is attached to the 5′ end of thesense strand of the dsRNA agent, the 3′ end of the antisense strand ofthe dsRNA agent, or the 5′ end of the antisense strand of the dsRNAagent.

In some embodiments, the dsRNA agents of the invention comprise aplurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independentlyattached to a plurality of nucleotides of the dsRNA agent through aplurality of monovalent linkers.

An exemplary dsRNA agent conjugated to the ligand as shown in thefollowing schematic:

and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the ligand is a cholesterol.

In certain embodiments, the region of complementarity comprises any oneof the antisense sequences in any one of Tables 3, 4, 6 and 7. Inanother embodiment, the region of complementarity consists of any one ofthe antisense sequences in any one of Tables 3, 4, 6 and 7.

In one embodiment, the dsRNA agent is selected from the group of dsRNAagents listed in any one of Tables 3, 4, 6, and 7.

In an aspect, the invention provides a double stranded ribonucleic acid(dsRNA) agents for inhibiting expression of a XDH gene, wherein thedsRNA agent comprises a sense strand and an antisense strand forming adouble stranded region, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1 or 9 and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:2 or 10,respectively, wherein substantially all of the nucleotides of the sensestrand comprise a nucleotide modification selected from a 2′-O-methylmodification and a 2′-fluoro modification, wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus, wherein substantially all of the nucleotides of theantisense strand comprise a nucleotide modification selected from a2′-O-methyl modification and a 2′-fluoro modification, wherein theantisense strand comprises two phosphorothioate internucleotide linkagesat the 5′-terminus and two phosphorothioate internucleotide linkages atthe 3′-terminus, and wherein the sense strand is conjugated to one ormore GalNAc derivatives attached through a branched bivalent ortrivalent linker at the 3′-terminus.

In certain embodiments, all of the nucleotides of the sense strand andall of the nucleotides of the antisense strand are modified nucleotides.In certain embodiments, each strand has 19-30 nucleotides.

In certain embodiments, substantially all of the nucleotides of thesense strand are modified. In certain embodiments, substantially all ofthe nucleotides of the antisense strand are modified. In certainembodiments, substantially all of the nucleotides of both the sensestrand and the antisense strand are modified.

In an aspect, the invention provides a cell containing the dsRNA agentas described herein.

In an aspect, the invention provides a vector encoding at least onestrand of a dsRNA agent, wherein the dsRNA agent comprises a region ofcomplementarity to at least a part of an mRNA encoding XDH, wherein thedsRNA agent is 30 base pairs or less in length, and wherein the dsRNAagent targets the mRNA for cleavage. In certain embodiments, the regionof complementarity is at least 15 nucleotides in length. In certainembodiments, the region of complementarity is 19 to 23 nucleotides inlength.

In an aspect, the invention provides a cell comprising a vector asdescribed herein.

In an aspect, the invention provides a pharmaceutical composition forinhibiting expression of an XDH gene comprising the dsRNA agent of theinvention. In one embodiment, the dsRNA agent is administered in anunbuffered solution. In certain embodiments, the unbuffered solution issaline or water. In other embodiments, the dsRNA agent is administeredwith a buffer solution. In such embodiments, the buffer solution cancomprises acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. For example, the buffer solution can be phosphatebuffered saline (PBS).

In an aspect, the invention provides a pharmaceutical compositioncomprising the dsRNA agent of the invention and a lipid formulation. Incertain embodiments, the lipid formulation comprises a LNP. In certainembodiments, the lipid formulation comprises a MC3.

In an aspect, the invention provides a method of inhibiting XDHexpression in a cell, the method comprising (a) contacting the cell withthe dsRNA agent of the invention or the pharmaceutical composition ofthe invention; and (b) maintaining the cell produced in step (a) for atime sufficient to obtain degradation of the mRNA transcript of an XDHgene, thereby inhibiting expression of the XDH gene in the cell. Incertain embodiments, the cell is within a subject, for example, a humansubject, for example a female human or a male human. In preferredembodiments, XDH expression is inhibited by at least 10%, 15%, 20%,preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or to belowthe threshold of detection.

In an aspect, the invention provides a method of treating a subjecthaving a disease or disorder that would benefit from reduction in XDHexpression, the method comprising administering to the subject atherapeutically effective amount of the dsRNA agent of the invention orthe pharmaceutical composition of the invention, thereby treating thesubject.

In an aspect, the invention provides a method of preventing at least onesymptom in a subject having a disease or disorder that would benefitfrom reduction in XDH expression, the method comprising administering tothe subject a prophylactically effective amount of the dsRNA agent ofthe invention or a pharmaceutical composition of the invention, therebypreventing at least one symptom in the subject having a disorder thatwould benefit from reduction in XDH expression.

In certain embodiments, the administration of the dsRNA agent to thesubject causes a decrease in the uric acid production. In certainembodiments, the administration of the dsRNA agent causes a decrease inthe level of XDH in the subject, e.g., the level of XO or XDH protein,or XDH mRNA in the subject.

In certain embodiments, the XDH-associated disease is gout. In certainembodiments, the XDH-associated disease is Lesch Nyhan syndrome. Inanother embodiment, the XDH-associated disease is glycogen storagedisease (GSD), e.g., GSD type Ia.

In certain embodiments, the invention further comprises administering anagent to decrease uric acid level in a subject, e.g., an inhibitor ofuric acid production or an agent that increases uric acid elimination,to the subject with an XDH-associated disease. Agents to decrease theuric acid level in a subject include, but are not limited to,allopurinol, febuxostat, or an interleukin-10 (IL-1) antagonist(canakinumab or rilonacept).

In certain embodiments, treatment of the subject with allopurinol iscontraindicated. For example, administration of allopurinol iscontraindicated in a subject with compromised renal function, due to,for example, chronic kidney disease, hypothyroidism, hyperinsulinemia,or insulin resistance. Administration of allopurinol may becontraindicated in combination with other drugs including, but notlimited to, oral coagulants and probenecid; subjects taking diureticsespecially thiazide diuretics or other drugs that can reduce kidneyfunction/have potential kidney toxicity.

In certain embodiments, the dsRNA agent is administered to a subjectwith an XDH-associated disease wherein the subject has compromised renalfunction. In certain embodiments, the dsRNA agent is administered to asubject with an XDH-associated disease wherein the subject ispredisposed to compromised renal function, e.g., in a subject withhypertension, metabolic disorder, Type 1 diabetes, Type 2 diabetes, orthe elderly. In certain embodiments, a subject is predisposed tocompromised renal function as a result of treatment with one or moreother therapeutic agents, e.g., diuretics.

In certain embodiments, the dsRNA agent is administered to a subjectwith an XDH-associated disease wherein the subject has failed treatmentwith allopurinol, e.g., gout flares during treatment, hypersensitivityreaction at any time after initiation of treatment with allopurinol orother unacceptable side effects as judged by the physician or patient.

Gout is often present in subjects who suffer from one or moreco-morbidities. In certain embodiments, the dsRNA agent is administeredto a subject with gout who has one or more of these co-morbidities. Forexample, in certain embodiments, the co-morbidity is reduced cardiacfunction or has cardiovascular disease. In certain embodiments, theco-morbidity is non-alcoholic fatty liver disease (NAFLD) ornon-alcoholic steatohepatitis (NASH). In certain embodiments, theco-morbidity is metabolic syndrome. In certain embodiments, theco-morbidity is hyperlipidemia. In certain embodiments, the co-morbidityis reduced renal function (e.g., glomerular filtration rate of less than60).

In certain embodiments if the invention, the dsRNA agent is administeredto a subject with gout who is not obese.

In various embodiments, the dsRNA agent is administered at a dose ofabout 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.In some embodiments, the dsRNA agent is administered at a dose of about10 mg/kg to about 30 mg/kg. In certain embodiments, the dsRNA agent isadministered at a dose selected from 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In certain embodiments, thedsRNA agent is administered about once per week, once per month, onceevery other two months, or once a quarter (i.e., once every threemonths) at a dose of about 0.1 mg/kg to about 5.0 mg/kg.

In certain embodiments, the dsRNA agent is administered to the subjectonce a week. In certain embodiments, the dsdsRNA agent is administeredto the subject once a month. In certain embodiments, the dsRNA agent isadministered once per quarter (i.e., every three months).

In some embodiment, the dsdsRNA agent is administered to the subjectsubcutaneously.

In various embodiments, the methods of the invention further comprisemonitoring the subject for at least one sign or symptom of anXDH-related disease, for example, gout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the uric acid metabolic pathway. XDH islabeled as XO in the schematic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a xanthine dehydrogenase (XDH) gene. The gene may bewithin a cell, e.g., a cell within a subject, such as a human. The useof these iRNAs enables the targeted degradation of mRNAs of thecorresponding gene (XDH gene) in mammals.

The iRNAs of the invention have been designed to target the human XDHgene, including portions of the gene that are conserved in the XDHorthologs of other mammalian species. Without intending to be limited bytheory, it is believed that the specific target sites or the specificmodifications in these iRNAs confer to the iRNAs of the inventionimproved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention also provides methods for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of an XDH gene, e.g., an XDH-associated disease, such asgout, using iRNA compositions which effect the RNA-induced silencingcomplex (RISC)-mediated cleavage of RNA transcripts of an XDH gene.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofan XDH gene. In certain embodiments, the iRNAs of the invention includean RNA strand (the antisense strand) which can include longer lengths,for example up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60,22-43, 27-53 nucleotides in length with a region of at least 19contiguous nucleotides that is substantially complementary to at least apart of an mRNA transcript of an XDH gene. These iRNAs with the longerlength antisense strands preferably include a second RNA strand (thesense strand) of 20-60 nucleotides in length wherein the sense andantisense strands form a duplex of 18-30 contiguous nucleotides.

The use of the iRNA agents described herein enable the targeteddegradation of mRNAs of the corresponding gene (XDH gene) in mammals.Very low dosages of the iRNAs of the invention, in particular, canspecifically and efficiently mediate RNA interference (RNAi), resultingin significant inhibition of expression of the corresponding gene (XDHgene). Using in vitro and in vivo assays, the present inventors havedemonstrated that iRNAs targeting an XDH gene can mediate RNAi,resulting in significant inhibition of expression of XDH and a decreasein uric acid levels. Thus, methods and compositions including theseiRNAs are useful for treating a subject having an XDH-associateddisease, such as gout.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of an XDH geneas well as compositions, uses, and methods for treating subjects havingdiseases and disorders that would benefit from reduction of theexpression of an XDH gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise. Forexample, “sense strand or antisense strand” is understood as “sensestrand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, aboutmeans±10%. In certain embodiments, about means±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understoodto include the number adjacent to the term “at least”, and allsubsequent numbers or integers that could logically be included, asclear from context. For example, the number of nucleotides in a nucleicacid molecule must be an integer. For example, “at least 18 nucleotidesof a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21nucleotides have the indicated property. When at least is present beforea series of numbers or a range, it is understood that “at least” canmodify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the valueadjacent to the phrase and logical lower values or integers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range.

As used herein, ranges include both the upper and lower limit.

In the event of a conflict between a sequence and its indicated site ona transcript or other sequence, the nucleotide sequence recited in thespecification takes precedence.

Various embodiments of the invention can be combined as determinedappropriate by one of skill in the art.

“Xanthine dehydrogenase” or “XDH” belongs to the group ofmolybdenum-containing hydroxylases involved in the oxidative metabolismof purines. The encoded protein has been identified as a moonlightingprotein based on its ability to perform mechanistically distinctfunctions. Xanthine dehydrogenase can be converted to xanthine oxidaseby reversible sulfhydryl oxidation or by irreversible proteolyticmodification. As used herein, unless clear from context, xanthinedehydrogenase or XDH is understood to include both the xanthinedehydrogenase and xanthine oxidase (“XO” or “XOR”) form of the protein.The protein is expressed predominantly in the intestine and the liver,but is also expressed in adipose tissue. Two transcript variants havebeen identified for the human isoform of the gene. Further informationon XDH is provided, for example in the NCBI Gene database atwww.ncbi.nlm.nih.gov/gene/7498 (which is incorporated herein byreference as of the date of filing this application). The amino acid andcomplete coding sequences of the reference sequence of the human XDHgene may be found in, for example, GenBank Accession No. GI: 91823270(RefSeq Accession No. NM_000379.3; SEQ ID NO:1; the reverse complementof SEQ ID NO:1 is shown in SEQ ID NO:2) and GenBank Accession No. GI:767915203 (RefSeq Accession No. XM_011533096; SEQ ID NO: 9; the reversecomplement of SEQ ID NO:9 is shown in SEQ ID NO:10). The nucleotide andamino acid sequence of mammalian orthologs of the human XDH gene may befound in, for example, GI: 575501724 (RefSeq Accession No. NM_011723.3,mouse; SEQ ID NO:3; the reverse complement of SEQ ID NO:3 is shown inSEQ ID NO:4); GI: 8394543 (RefSeq Accession No. NM_017154.1, rat; SEQ IDNO:5; the reverse complement of SEQ ID NO:5 is shown in SEQ ID NO:6);GenBank Accession Nos. GI: 544482046 (RefSeq Accession Nos.XM_005576183.1 and XM_005576184.1, cynomolgus monkey; SEQ ID NO:7; thereverse complement of SEQ ID NO:7 is shown in SEQ ID NO:8, and SEQ IDNO:15; the reverse complement of SEQ ID NO:15 is shown in SEQ ID NO:16,respectively).

A number of naturally occurring SNPs in the XDH gene are known and canbe found, for example, in the SNP database at the NCBI athttp://www.ncbi.nlm.nih.gov/snp?LinkName=gene_snp&from_uid=7498 (whichis incorporated herein by reference as of the date of filing thisapplication) which lists over 3000 SNPs in human XDH. In preferredembodiments, such naturally occuring variants are included within thescope of the XDH gene sequence.

Additional examples of XDH mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an XDH gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target portion of the sequence willbe at least long enough to serve as a substrate for iRNA-directedcleavage at or near that portion of the nucleotide sequence of an mRNAmolecule formed during the transcription of an XDH gene. In oneembodiment, the target sequence is within the protein coding region ofXDH.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine, and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” “iRNA agent,” “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of an XDH gene in a cell, e.g., a cell within a subject,such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNAi that interacts with a target RNA sequence, e.g., an XDHtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into double stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processesthese dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the invention relates to a singlestranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generatedwithin a cell and which promotes the formation of a RISC complex toeffect silencing of the target gene, i.e., an XDH gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides in length and are chemically modified. Thedesign and testing of single-stranded RNAs are described in U.S. Pat.No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an XDH gene. In some embodiments ofthe invention, a double stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide or a modified nucleotide. In addition, as used inthis specification, an “iRNA” may include ribonucleotides with chemicalmodifications; an iRNA may include substantial modifications at multiplenucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, or modified nucleobase, or any combinationthereof. Thus, the term modified nucleotide encompasses substitutions,additions or removal of, e.g., a functional group or atom, tointernucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“iRNA” or “RNAi agent” for the purposes of this specification andclaims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments,the hairpin loop can be 10 or fewer nucleotides. In some embodiments,the hairpin loop can be 8 or fewer unpaired nucleotides. In someembodiments, the hairpin loop can be 4-10 unpaired nucleotides. In someembodiments, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In certain embodiments, an iRNA agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an XDH gene. Without wishing to be bound bytheory, long double stranded RNA introduced into cells is broken downinto siRNA by a Type III endonuclease known as Dicer (Sharp et al.(2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme,processes the dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). The siRNAs are then incorporated into an RNA-induced silencingcomplex (RISC) where one or more helicases unwind the siRNA duplex,enabling the complementary antisense strand to guide target recognition(Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriatetarget mRNA, one or more endonucleases within the RISC cleave the targetto induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In some embodiments, an iRNA of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., an XDHtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory, long double stranded RNA introduced intocells is broken down into siRNA by a Type III endonuclease known asDicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, aribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs(Bernstein, et al., (2001) Nature 409:363). The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of a doublestranded iRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15nucleotides. In certain embodiments, the overhang can be at least threenucleotides, at least four nucleotides, at least five nucleotides ormore. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand, or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of either an antisense orsense strand of a dsRNA.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1-3, 1-4, 2-4, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotide, overhang at the 3′-end or the 5′-end. In certainembodiments, the overhang on the sense strand or the antisense strand,or both, can include extended lengths longer than 10 nucleotides, e.g.,1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15nucleotides in length. In certain embodiments, an extended overhang ison the sense strand of the duplex. In certain embodiments, an extendedoverhang is present on the 3′end of the sense strand of the duplex. Incertain embodiments, an extended overhang is present on the 5′end of thesense strand of the duplex. In certain embodiments, an extended overhangis on the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 3′end of the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the5′end of the antisense strand of the duplex. In certain embodiments, oneor more of the nucleotides in the overhang is replaced with a nucleosidethiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” double stranded RNAi agent is double strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withno nucleotide overhang at one end (i.e., agents with one overhang andone blunt end) or with no nucleotide overhangs at either end.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an XDH mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., an XDH nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, adouble stranded RNAi agent of the invention includes a nucleotidemismatch in the antisense strand. In some embodiments, a double strandedRNAi agent of the invention includes a nucleotide mismatch in the sensestrand. In some embodiments, the nucleotide mismatch is, for example,within 5, 4, 3, 2, or 1 nucleotides from the 3′-end of the iRNA. Inanother embodiment, the nucleotide mismatch is, for example, in the3′-terminal nucleotide of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, “substantially all of the nucleotides are modified” arelargely but not wholly modified and can include not more than 5, 4, 3,2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs or base pairs formedfrom non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary,” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a double stranded RNAi agent and a targetsequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding an XDH gene). For example, apolynucleotide is complementary to at least a part of an XDH mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding an XDH gene.

Accordingly, in some embodiments, the antisense polynucleotidesdisclosed herein are fully complementary to the target XDH sequence. Insome embodiments, the sense polynucleotides disclosed herein are fullycomplementary to the antisense sequence of a target XDH sequence.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of the nucleotidesequence of SEQ ID NO:1 or 2, or a fragment of SEQ ID NO:1 or 2, such asabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about% 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% complementary.

In other embodiments, the sense polynucleotides disclosed herein aresubstantially complementary to the antisense sequence of a target XDHsequence and comprise a contiguous nucleotide sequence which is at leastabout 80% complementary over its entire length to the equivalent regionof the nucleotide sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2,such as about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target XDH sequenceand comprises a contiguous nucleotide sequence which is at least about80% complementary over its entire length to any one of the sense strandnucleotide sequences in any one of Tables 3, 4, 6 and 7, or a fragmentof any one of the sense strand nucleotide sequences in any one of Tables3, 4, 6 and 7, such as about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, an iRNA of the invention includes an antisensestrand that is substantially complementary to the target XDH sequenceand comprises a contiguous nucleotide sequence which is at least about80% complementary over its entire length to the equivalent region of thenucleotide sequence of any one of the antisense strand nucleotidesequences in any one of Tables 3, 4, 6 and 7, or a fragment of any oneof the antisense strand nucleotide sequences in any one of Tables 3, 4,6 and 7, such as at least 85%, 90%, 95% complementary, or 100%complementary.

In an aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisenseoligonucleotide molecule that inhibits a target mRNA via an antisenseinhibition mechanism. The single-stranded antisense oligonucleotidemolecule is complementary to a sequence within the target mRNA. Thesingle-stranded antisense oligonucleotides can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The single-stranded antisense oligonucleotidemolecule may be about 14 to about 30 nucleotides in length and have asequence that is complementary to a target sequence. For example, thesingle-stranded antisense oligonucleotide molecule may comprise asequence that is at least about 14, 15, 16, 17, 18, 19, 20, or morecontiguous nucleotides from any one of the antisense sequences describedherein.

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as usedherein, includes contacting a cell by any possible means. Contacting acell with an iRNA includes contacting a cell in vitro with the iRNA orcontacting a cell in vivo with the iRNA. The contacting may be donedirectly or indirectly. Thus, for example, the iRNA may be put intophysical contact with the cell by the individual performing the method,or alternatively, the iRNA may be put into a situation that will permitor cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the iRNA. Contacting a cell in vivo may be done, for example,by injecting the iRNA into or near the tissue where the cell is located,or by injecting the iRNA into another area, e.g., the bloodstream or thesubcutaneous space, such that the agent will subsequently reach thetissue where the cell to be contacted is located. For example, the iRNAmay contain or be coupled to a ligand, e.g., GalNAc3, that directs theiRNA to a site of interest, e.g., the liver. Combinations of in vitroand in vivo methods of contacting are also possible. For example, a cellmay also be contacted in vitro with an iRNA and subsequentlytransplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes“introducing” or “delivering the iRNA into the cell” by facilitating oreffecting uptake or absorption into the cell. Absorption or uptake of aniRNA can occur through unaided diffusion or active cellular processes,or by auxiliary agents or devices. Introducing an iRNA into a cell maybe in vitro or in vivo. For example, for in vivo introduction, iRNA canbe injected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and USPublication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or are knownin the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, and a horse), or a bird (e.g., a duck or a goose) thatexpresses the target gene, either endogenously or heterologously. Incertain embodiments, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in XDH gene expression or replication; a human atrisk for a disease, disorder or condition that would benefit fromreduction in XDH gene expression; a human having a disease, disorder orcondition that would benefit from reduction in XDH gene expression; orhuman being treated for a disease, disorder or condition that wouldbenefit from reduction in XDH gene expression, as described herein. Insome embodiments, the subject is a female human. In other embodiments,the subject is a male human.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with XDH geneexpression or XDH protein production, e.g., gout, NASH, or NAFLD.“Treatment” can also mean prolonging survival as compared to expectedsurvival in the absence of treatment.

The term “lower” in the context of the level of XDH gene expression orXDH protein production in a subject, or a disease marker or symptomrefers to a statistically significant decrease in such level. Thedecrease can be, for example, at least 20%, 25%, preferably at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or below the level of detection for the detection method. In certainembodiments, the expression of the target is normalized, i.e., decreasedto a level accepted as within the range of normal for an individualwithout such disorder. For example, chronic hyperuricemia is defined asserum urate levels greater than 6.8 mg/dl (greater than 360 mmol/), thelevel above which the physiological saturation threshold is exceeded(Mandell, Cleve. Clin. Med. 75:S5-S8, 2008). In certain embodiments, thereduction is the normalization of the level of a sign or symptom of adisease, a reduction in the difference between the subject level of asign of the disease and the normal level of the sign for the disease(e.g., the upper level of normal when the level must be reduced to reacha normal level, and the lower level of normal when the level must beincreased to reach a normal level). In certain embodiments, the methodsinclude a clinically relevant inhibition of expression of XDH, e.g., asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of XDH.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder, or condition, that would benefit from a reductionin expression of an XDH gene or production of XDH protein, i.e., adisease, disorder, or condition that would benefit from reduction of achronically elevated uric acid level, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., a symptom of XDH gene expression,such as the presence of chronic elevated serum levels of uric acid,e.g., hyperuricemia, gout, NASH, NAFLD, metabolic disorder, orcardiovascular disease. The failure to develop a disease, disorder orcondition, or the reduction in the development of a symptom orcomorbidity associated with such a disease, disorder or condition (e.g.,by at least about 10% on a clinically accepted scale for that disease ordisorder), the exhibition of delayed symptoms or disease progression bydays, weeks, months or years, or the reduction or maintenance of a serumuric acid level at 6.8 mg/dl or less in a subject prone to elevatedserum uric acid is considered effective prevention. Prevention mayrequire the administration of more than one dose.

As used herein, the term “xanthine dehydrogenase-associated disease” or“XDH-associated disease,” is a disease or disorder that is caused by, orassociated with XDH gene expression or XDH protein production, e.g., adisease, disorder, or condition associated with chronic elevated levelsof serum uric acid. The term “XDH-associated disease” includes adisease, disorder, or condition that would benefit from a decrease inXDH gene expression, replication, or protein activity. Non-limitingexamples of XDH-associated diseases include, for example, hyperuricemia,gout, NAFLD, NASH, metabolic disorder, insulin resistance,cardiovascular disease, type 2 diabetes, and conditions linked tooxidative stress e.g., chronic low grade inflammation; or otherXDH-associated disease.

In certain embodiments, an XDH-associated disease is gout. In certainembodiments, an XDH-associated disease is NASH or NAFLD.

As used herein, “chronic renal insufficiency” or “chronic kidneydisease” is a condition characterized by a gradual loss of kidneyfunction over time. Chronic kidney disease is commonly caused by highblood pressure or diabetes, but may result from other diseases orconditions such as lupus or may be caused by medications that have renaltoxicity.

“Glomerulonephritis” includes a group of diseases that causeinflammation and damage to the kidney's filtering units. Inheriteddiseases, such as polycystic kidney disease, which causes large cysts toform in the kidneys and damage the surrounding tissue.

Congenital structural malformation of the kidneys, kidney stones orother obstructions (e.g., enlarged prostate) can result in chronickidney disease.

Without being bound by mechanism, reduced kidney function can result inimpaired renal clearance of uric acid. Kidney function is typicallyassessed by glomerular filtration rate (GFR) which is calculated basedon blood creatinine level, age, body size, and gender. A lower GFR isindicative of lower kidney function. Kidney disease is typicallydiagnosed with a GFR of less than 60 for at least three months or a GFRof 60 or less with high protein in the urine is diagnostic of chronickidney disease. However, a GFR of less than 90 is indicative of kidneydamage with mild loss of kidney function.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a patient fortreating a subject having hyperuricemia, gout, NAFLD, NASH, metabolicdisorder, insulin resistance, cardiovascular disease, type 2 diabetes,and conditions linked to oxidative stress, e.g., chronic low gradeinflammation; or other XDH-associated disease, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease orits related comorbidities). The “therapeutically effective amount” mayvary depending on the iRNA, how it is administered, the disease and itsseverity and the history, age, weight, family history, genetic makeup,stage of pathological processes mediated by XDH gene expression, thetypes of preceding or concomitant treatments, if any, and otherindividual characteristics of the patient to be treated. Treatment mayrequire the administration of more than one dose.

As used herein, a therapeutically effective amount is sufficient toresult in a clinically relevant lowering of chronic serum uric acidlevel in a subject and does not necessarily require the lowering of thechronic serum uric acid level to below 6.8 mg/dl. In certainembodiments, a therapeutically effective amount of an iRNA will lowerthe chronic uric acid of a subject to 6.8 mg/dl of uric acid or less,e.g., to 6 mg/dl or less, and optionally no lower than about 2 mg/dl.Studies of subjects with inherited disorders associated with profound,lifelong hypouricemia indicate that maintaining serum uric acid near orbelow 2 mg/dl is safe (Hershfeld, Curr. Opin. Rheumatol. 21:138-142,2009).

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subject whodoes not yet experience or display symptoms of hyperuricemia, gout,NAFLD, NASH, metabolic disorder, insulin resistance, cardiovasculardisease, type 2 diabetes, and conditions linked to oxidative stress,e.g., chronic low grade inflammation; or other XDH-associated disease,but who may be predisposed to an XDH-associated disease, is sufficientto prevent or delay the development or progression of the disease or oneor more symptoms of the disease for a clinically significant period oftime, e.g., to lower a chronically elevated serum uric acid level, toprevent an increase in chronic serum uric acid level or to maintain auric acid level below 6.8 mg/dl in a subject prone to elevated chronicserum uric acid level. The “prophylactically effective amount” may varydepending on the iRNA, how it is administered, the degree of risk ofdisease, and the history, age, weight, family history, genetic makeup,the types of preceding or concomitant treatments, if any, and otherindividual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an iRNA that produces some desiredlocal or systemic effect at a reasonable benefit/risk ratio applicableto any treatment. iRNAs employed in the methods of the present inventionmay be administered in a sufficient amount to produce a reasonablebenefit/risk ratio applicable to such treatment. For example, in certainembodiments, treatment with the iRNAs of the invention will result inserum uric acid levels at 2-6.8 mg/dl, preferably 2-6 mg/dl in subjects.Maintenance of such uric acid levels will treat or preventXDH-associated diseases.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates or polyanhydrides;(22) bulking agents, such as polypeptides and amino acids (23) serumcomponent, such as serum albumin, HDL and LDL; and (22) other non-toxiccompatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs, or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). A “sample derived from a subject” can refer to blood drawnfrom the subject or plasma derived therefrom.

I. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of anXDH gene. In preferred embodiments, the iRNA is a double strandedribonucleic acid (dsRNA) molecule for inhibiting the expression of anXDH gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having an XDH-associated disease, e.g., hyperuricemia,gout. The dsRNA agent includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an XDH gene. The region of complementarityis about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length).Upon contact with a cell expressing the XDH gene, the iRNA inhibits theexpression of the XDH gene (e.g., a human, a primate, a non-primate, ora bird XDH gene) by at least about 20% as assayed by, for example, a PCRor branched DNA (bDNA)-based method, or by a protein-based method, suchas by immunofluorescence analysis, using, for example, western blottingor flow cytometric techniques. In preferred embodiments, inhibition ofexpression is determined by the qPCR method provided in the examples.For in vitro assessment of activity, percent inhibition is determinedusing the methods provided in Example 2 at a single dose at a 10 nMduplex final concentration. For in vivo studies, the level aftertreatment can be compared to, for example, an appropriate historicalcontrol or a pooled population sample control to determine the level ofreduction, e.g., when a baseline value is not available for the subject.

A dsRNA agent includes two RNA strands that are complementary andhybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence. The target sequencecan be derived from the sequence of an mRNA formed during the expressionof an XDH gene. The other strand (the sense strand) includes a regionthat is complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g.,15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20,15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24,18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26,21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengthsintermediate to the above recited ranges and lengths are alsocontemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence may be15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

In some embodiments, the dsRNA is about 15 to 23 nucleotides in length,or about 25 to 30 nucleotides in length. In general, the dsRNA is longenough to serve as a substrate for the Dicer enzyme. For example, it iswell-known in the art that dsRNAs longer than about 21-23 nucleotides inlength may serve as substrates for Dicer. As the ordinarily skilledperson will also recognize, the region of an RNA targeted for cleavagewill most often be part of a larger RNA molecule, often an mRNAmolecule. Where relevant, a “part” of an mRNA target is a contiguoussequence of an mRNA target of sufficient length to allow it to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to about 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36,15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34,11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33,14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31,10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27,15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17,18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21,18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22,19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23,20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or21-22 base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target XDH gene expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3,or 4 nucleotides. dsRNAs having at least one nucleotide overhang canhave superior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand, or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of an antisense or sensestrand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

Double stranded RNAi compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double strandedRNA molecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Similarly, single-stranded oligonucleotides of the invention can beprepared using solution-phase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an antisense sequence. The sense strandis selected from the group of sequences provided in any one of Tables 3,4, 6 and 7, and the corresponding antisense strand of the sense strandis selected from the group of sequences of any one of Tables 3, 4, 6 and7. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of an XDH gene. As such, in this aspect, a dsRNA will includetwo oligonucleotides, where one oligonucleotide is described as thesense strand in any one of Tables 3, 4, 6 and 7, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Tables 3, 4, 6 and 7. In certainembodiments, the substantially complementary sequences of the dsRNA arecontained on separate oligonucleotides. In other embodiments, thesubstantially complementary sequences of the dsRNA are contained on asingle oligonucleotide.

It will be understood that, although some of the sequences in Tables 3,4, 6 and 7 are described as modified and/or conjugated sequences, theRNA of the iRNA of the invention e.g., a dsRNA of the invention, maycomprise any one of the sequences set forth in Tables 3, 4, 6 and 7 thatis un-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

The skilled person is well aware that dsRNAs having a duplex structureof about 20 to 23 base pairs, e.g., 21, base pairs have been hailed asparticularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can also be effective (Chu and Rana (2007)RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3, 4, 6 and 7,dsRNAs described herein can include at least one strand of a length ofminimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of Tables 3, 4, 6 and 7 minus onlya few nucleotides on one or both ends can be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs having a sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotidesderived from one of the sequences of Tables 3, 4, 6 and 7, and differingin their ability to inhibit the expression of an XDH gene by not morethan about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprisingthe full sequence, are contemplated to be within the scope of thepresent invention.

In addition, the RNAs provided in Tables 3, 4, 6 and 7 identify asite(s) in an XDH transcript that is susceptible to RISC-mediatedcleavage. As such, the present invention further features iRNAs thattarget within one of these sites. As used herein, an iRNA is said totarget within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in Tables 3, 4, 6 and 7coupled to additional nucleotide sequences taken from the regioncontiguous to the selected sequence in an XDH gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art or provided herein) to identify thosesequences that perform optimally can identify those RNA sequences that,when targeted with an iRNA agent, mediate the best inhibition of targetgene expression. Thus, while the sequences identified, for example, inTables 3, 4, 6 and 7 represent effective target sequences, it iscontemplated that further optimization of inhibition efficiency can beachieved by progressively “walking the window” one nucleotide upstreamor downstream of the given sequences to identify sequences with equal orbetter inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3, 4, 6 and 7, further optimization could be achieved bysystematically either adding or removing nucleotides to generate longeror shorter sequences and testing those sequences generated by walking awindow of the longer or shorter size up or down the target RNA from thatpoint. Again, coupling this approach to generating new candidate targetswith testing for effectiveness of iRNAs based on those target sequencesin an inhibition assay as known in the art or as described herein canlead to further improvements in the efficiency of inhibition. Furtherstill, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in overhang, or other modifications asknown in the art or discussed herein to further optimize the molecule(e.g., increasing serum stability or circulating half-life, increasingthermal stability, enhancing transmembrane delivery, targeting to aparticular location or cell type, increasing interaction with silencingpathway enzymes, increasing release from endosomes) as an expressioninhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of an XDH gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of an XDH gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of an XDHgene is important, especially if the particular region ofcomplementarity in an XDH gene is known to have polymorphic sequencevariation within the population.

II. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., adsRNA, is un-modified, and does not comprise, e.g., chemicalmodifications or conjugations known in the art and described herein. Inother embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA,is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA orsubstantially all of the nucleotides of an iRNA are modified, i.e., notmore than 5, 4, 3, 2, or 1 unmodified nucleotides are present in astrand of the iRNA.

The nucleic acids featured in the invention can be synthesized ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; or backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative US patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts.

Representative US patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein,in which both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with novel groups. Thebase units are maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound in which an RNAmimetic that has been shown to have excellent hybridization propertiesis referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative US patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents ofeach of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative US patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxy-thymine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative US patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)-0-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′(see, e.g., US Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative US patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

In some embodiments, the iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative US publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an iRNA.Suitable phosphate mimics are disclosed in, for example US PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in U.S. Patent Publication No. 2014/0315835 and PCTPublication No. WO 2013/075035, the entire contents of each of which areincorporated herein by reference. WO 2013/075035 and U.S. 2014/0315835provide motifs of three identical modifications on three consecutivenucleotides into a sense strand or antisense strand of a dsRNAi agent,particularly at or near the cleavage site. In some embodiments, thesense strand and antisense strand of the dsRNAi agent may otherwise becompletely modified. The introduction of these motifs interrupts themodification pattern, if present, of the sense or antisense strand. ThedsRNAi agent may be optionally conjugated with a GalNAc derivativeligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of thedouble stranded RNAi agent are completely modified to have one or moremotifs of three identical modifications on three consecutive nucleotidesat or near the cleavage site of at least one strand of a dsRNAi agent,the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., XDH gene) in vivo.The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may be, independently, 12-30 nucleotides inlength. For example, each strand may independently be 14-30 nucleotidesin length, 17-30 nucleotides in length, 25-30 nucleotides in length,27-30 nucleotides in length, 17-23 nucleotides in length, 17-21nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides inlength, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” Theduplex region of a dsRNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be 14-30 nucleotide pairs in length,17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length,17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length,17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length,19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length,21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.In another example, the duplex region is selected from 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides inlength.

In certain embodiments, the dsRNAi agent may contain one or moreoverhang regions or capping groups at the 3′-end, 5′-end, or both endsof one or both strands. The overhang can be, independently, 1-6nucleotides in length, for instance 2-6 nucleotides in length, 1-5nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides inlength, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3nucleotides in length, or 1-2 nucleotides in length. In certainembodiments, the overhang regions can include extended overhang regionsas provided above. The overhangs can be the result of one strand beinglonger than the other, or the result of two strands of the same lengthbeing staggered. The overhang can form a mismatch with the target mRNAor it can be complementary to the gene sequences being targeted or canbe another sequence. The first and second strands can also be joined,e.g., by additional bases to form a hairpin, or by other non-baselinkers.

In certain embodiments, the nucleotides in the overhang region of thedsRNAi agent can each independently be a modified or unmodifiednucleotide including, but no limited to 2′-sugar modified, such as,2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine(Teo), 2′-O-methoxyethyladenosine (Aeo),2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof. For example, TT can be an overhang sequence for either end oneither strand. The overhang can form a mismatch with the target mRNA orit can be complementary to the gene sequences being targeted or can beanother sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or bothstrands of the dsRNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In some embodiments, the overhang ispresent at the 3′-end of the sense strand, antisense strand, or bothstrands. In some embodiments, this 3′-overhang is present in theantisense strand. In some embodiments, this 3′-overhang is present inthe sense strand.

The dsRNAi agent may contain only a single overhang, which canstrengthen the interference activity of the RNAi, without affecting itsoverall stability. For example, the single-stranded overhang may belocated at the 3′-end of the sense strand or, alternatively, at the3-end of the antisense strand. The RNAi may also have a blunt end,located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of thedsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. While not wishing to be bound by theory, the asymmetric blunt endat the 5′-end of the antisense strand and 3′-end overhang of theantisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of19 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sensestrand and a 23 nucleotide antisense strand, wherein the sense strandcontains at least one motif of three 2′-F modifications on threeconsecutive nucleotides at positions 9, 10, 11 from the 5′end; theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at positions 11, 12, 13from the 5′end, wherein one end of the RNAi agent is blunt, while theother end comprises a 2 nucleotide overhang. Preferably, the 2nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In certain embodiments, everynucleotide in the sense strand and the antisense strand of the dsRNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In certain embodiments each residue isindependently modified with a 2′-O-methyl or 3′-fluoro, e.g., in analternating motif. Optionally, the dsRNAi agent further comprises aligand (preferably GalNAc, e.g., GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and anantisense strand, wherein the sense strand is 25-30 nucleotide residuesin length, wherein starting from the 5′ terminal nucleotide (position 1)positions 1 to 23 of the first strand comprise at least 8ribonucleotides; the antisense strand is 36-66 nucleotide residues inlength and, starting from the 3′ terminal nucleotide, comprises at least8 ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In certain embodiments, the dsRNAi agent comprises sense and antisensestrands, wherein the dsRNAi agent comprises a first strand having alength which is at least 25 and at most 29 nucleotides and a secondstrand having a length which is at most 30 nucleotides with at least onemotif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ endof the first strand and the 5′ end of the second strand form a blunt endand the second strand is 1-4 nucleotides longer at its 3′ end than thefirst strand, wherein the duplex region region which is at least 25nucleotides in length, and the second strand is sufficientlycomplemenatary to a target mRNA along at least 19 nucleotide of thesecond strand length to reduce target gene expression when the RNAiagent is introduced into a mammalian cell, and wherein Dicer cleavage ofthe dsRNAi agent preferentially results in an siRNA comprising the3′-end of the second strand, thereby reducing expression of the targetgene in the mammal. Optionally, the dsRNAi agent further comprises aligand.

In certain embodiments, the sense strand of the dsRNAi agent contains atleast one motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In certain embodiments, the antisense strand of the dsRNAi agent canalso contain at least one motif of three identical modifications onthree consecutive nucleotides, where one of the motifs occurs at or nearthe cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 17-23 nucleotides inlength, the cleavage site of the antisense strand is typically aroundthe 10, 11, and 12 positions from the 5′-end. Thus the motifs of threeidentical modifications may occur at the 9, 10, 11 positions; the 10,11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; orthe 13, 14, 15 positions of the antisense strand, the count startingfrom the first nucleotide from the 5′-end of the antisense strand, or,the count starting from the first paired nucleotide within the duplexregion from the 5′-end of the antisense strand. The cleavage site in theantisense strand may also change according to the length of the duplexregion of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may containmore than one motif of three identical modifications on threeconsecutive nucleotides. The first motif may occur at or near thecleavage site of the strand and the other motifs may be a wingmodification. The term “wing modification” herein refers to a motifoccurring at another portion of the strand that is separated from themotif at or near the cleavage site of the same strand. The wingmodification is either adjacent to the first motif or is separated by atleast one or more nucleotides. When the motifs are immediately adjacentto each other then the chemistries of the motifs are distinct from eachother, and when the motifs are separated by one or more nucleotide thanthe chemistries can be the same or different. Two or more wingmodifications may be present. For instance, when two wing modificationsare present, each wing modification may occur at one end relative to thefirst motif which is at or near cleavage site or on either side of thelead motif.

Like the sense strand, the antisense strand of the dsRNAi agent maycontain more than one motif of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two terminal nucleotides at the 3′-end, 5′-end, or bothends of the strand.

In other embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two paired nucleotides within the duplex region at the3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two,or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two, or three nucleotides; two modifications each from one strand fallon the other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two, or three nucleotides in theduplex region.

In some embodiments, every nucleotide in the sense strand and antisensestrand of the dsRNAi agent, including the nucleotides that are part ofthe motifs, may be modified. Each nucleotide may be modified with thesame or different modification which can include one or more alterationof one or both of the non-linking phosphate oxygens or of one or more ofthe linking phosphate oxygens; alteration of a constituent of the ribosesugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′- or 5′-terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a dsRNAi agent or may only occur in a single strandregion of a dsRNAi agent. For example, a phosphorothioate modificationat a non-linking O position may only occur at one or both ends, may onlyoccur in a terminal region, e.g., at a position on a terminalnucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, ormay occur in double strand and single strand regions, particularly atthe ends. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang,or in both. For example, it can be desirable to include purinenucleotides in overhangs. In some embodiments all or some of the basesin a 3′- or 5′-overhang may be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ position of the ribose sugar with modificationsthat are known in the art, e.g., the use of deoxyribonucleotides,2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of theribosugar of the nucleobase, and modifications in the phosphate group,e.g., phosphorothioate modifications. Overhangs need not be homologouswith the target sequence.

In some embodiments, each residue of the sense strand and antisensestrand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In some embodiments, the dsRNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′ to 3′ of the strand and the alternating motif inthe antisense strand may start with “BABABA” from 5′ to 3′ of the strandwithin the duplex region. As another example, the alternating motif inthe sense strand may start with “AABBAABB” from 5′ to 3′ of the strandand the alternating motif in the antisenese strand may start with“BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so thatthere is a complete or partial shift of the modification patternsbetween the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand or antisense strandinterrupts the initial modification pattern present in the sense strandor antisense strand. This interruption of the modification pattern ofthe sense or antisense strand by introducing one or more motifs of threeidentical modifications on three consecutive nucleotides to the sense orantisense strand may enhance the gene silencing activity against thetarget gene.

In some embodiments, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)or N_(b) may be present or absent when there is a wing modificationpresent.

The iRNA may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand, antisense strand, or both strands in anyposition of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand or antisense strand; or thesense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-standed RNAi agent comprises 6-8phosphorothioateinternucleotide linkages. In some embodiments, the antisense strandcomprises two phosphorothioate internucleotide linkages at the 5′-endand two phosphorothioate internucleotide linkages at the 3′-end, and thesense strand comprises at least two phosphorothioate internucleotidelinkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, or the 5′end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, thedsRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mistmatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of thefirst 1, 2, 3, 4, or 5 base pairs within the duplex regions from the5′-end of the antisense strand independently selected from the group of:A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other thancanonical pairings or pairings which include a universal base, topromote the dissociation of the antisense strand at the 5′-end of theduplex.

In certain embodiments, the nucleotide at the 1 position within theduplex region from the 5′-end in the antisense strand is selected fromA, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or3 base pair within the duplex region from the 5′-end of the antisensestrand is an AU base pair. For example, the first base pair within theduplex region from the 5′-end of the antisense strand is an AU basepair.

In other embodiments, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT) or the nucleotide at the 3′-end of the antisensestrand is deoxy-thymine (dT). For example, there is a short sequence ofdeoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-endof the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented byformula (I):

(I)5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications ofalternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage siteof the sense strand. For example, when the dsRNAi agent has a duplexregion of 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7,8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of the sensestrand, the count starting from the first nucleotide, from the 5′-end;or optionally, the count starting at the first paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

(Ib) 5′n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′; (Ic)5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′;  or (Id)5′n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-ZZZ-N_(a)-n_(q) 3′

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2, or 0 modified nucleotides. Each N_(a) can independently representan oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, N_(b) is 0,1, 2, 3, 4, 5, or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

(Ia) 5′n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′.

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

(II)5′n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(i)-N′_(a)-n_(p)′3′

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNAi agent has a duplex region of 17-23nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10,11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the first nucleotide, from the5′-end; or optionally, the count starting at the first paired nucleotidewithin the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motifoccurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and 1 is 0, or k is 0 and 1 is 1, or bothk and 1 are 1.

The antisense strand can therefore be represented by the followingformulas:

(IIb) 5′n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p) 3′; (IIc)5′n_(a)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p) 3′; or (IId)5′n_(q)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p) 3′

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5, or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

(Ia) 5′n_(p)-N_(a)′-Y′Y′Y′-N_(a)′-n_(q) 3′

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may containYYY motif occurring at 9, 10, and 11 positions of the strand when theduplex region is 21 nt, the count starting from the first nucleotidefrom the 5′-end, or optionally, the count starting at the first pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe first nucleotide from the 5′-end, or optionally, the count startingat the first paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the dsRNAi agents for use in the methods of the inventionmay comprise a sense strand and an antisense strand, each strand having14 to 30 nucleotides, the iRNA duplex represented by formula (III):

(III) sense:5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)N_(a)-n_(q) 3′antisense:3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(i)-N_(a)-n_(q) 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1;or both k and 1 are 0; or both k and 1 are 1.

Exemplary combinations of the sense strand and antisense strand formingan iRNA duplex include the formulas below:

(IIIa) 5′ n_(p)-N_(a)-Y Y Y -N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIb)5′ n_(p)-N_(a)-Y Y Y -N_(b)-Z Z Z -N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIc)5′ n_(p)-N_(a)-X X X -N_(b)-Y Y Y -N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIId)5′ n_(p)-N_(a)-X X X -N_(b)-Y Y Y -N_(b)-Z Z Z -N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′

When the dsRNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. EachN_(a), N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b), and N_(b)′ independently comprises modifications ofalternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and(IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide isdifferent than the modification on the Y′ nucleotide, the modificationon the Z nucleotide is different than the modification on the Z′nucleotide, or the modification on the X nucleotide is different thanthe modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications. In other embodiments, when the RNAi agent is representedby formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet otherembodiments, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In other embodiments, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula(IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications, n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via phosphorothioate linkage, the sense strandcomprises at least one phosphorothioate linkage, and the sense strand isconjugated to one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at leasttwo duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In some embodiments, the dsRNAi agent is a multimer containing three,four, five, six, or more duplexes represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId), wherein the duplexes are connected by alinker. The linker can be cleavable or non-cleavable. Optionally, themultimer further comprises a ligand. Each of the duplexes can target thesame gene or two different genes; or each of the duplexes can targetsame gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one offormulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to eachother at the 5′ end, and one or both of the 3′ ends, and are optionallyconjugated to a ligand. Each of the agents can target the same gene ortwo different genes; or each of the agents can target same gene at twodifferent target sites.

Various publications describe multimeric iRNAs that can be used in themethods of the invention. Such publications include WO2007/091269, U.S.Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the iRNA that contains conjugationsof one or more carbohydrate moieties to an iRNA can optimize one or moreproperties of the iRNA. In many cases, the carbohydrate moiety will beattached to a modified subunit of the iRNA. For example, the ribosesugar of one or more ribonucleotide subunits of a iRNA can be replacedwith another moiety, e.g., a non-carbohydrate (preferably cyclic)carrier to which is attached a carbohydrate ligand. A ribonucleotidesubunit in which the ribose sugar of the subunit has been so replaced isreferred to herein as a ribose replacement modification subunit (RRMS).A cyclic carrier may be a carbocyclic ring system, i.e., all ring atomsare carbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, anddecalin; preferably, the acyclic group is a serinol backbone ordiethanolamine backbone.

In certain embodiments, the iRNA for use in the methods of the inventionis an agent selected from agents listed in Table 3, Table 4, Table 6 orTable 7. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the iRNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the iRNA e.g., into a cell. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid(Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), athioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad.Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands do nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, galactose, transferrin, bisphosphonate,polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bileacid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGDpeptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, or intermediate filaments. The drug can be, for example,taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases, or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the useof an oligonucleotide that bears a pendant reactive functionality, suchas that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive oligonucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid orlipid-based molecule. Such a lipid or lipid-based molecule preferablybinds a serum protein, e.g., human serum albumin (HSA). An HSA bindingligand allows for distribution of the conjugate to a target tissue,e.g., a non-kidney target tissue of the body. For example, the targettissue can be the liver, including parenchymal cells of the liver. Othermolecules that can bind HSA can also be used as ligands. For example,naproxen or aspirin can be used. A lipid or lipid-based ligand can (a)increase resistance to degradation of the conjugate, (b) increasetargeting or transport into a target cell or cell membrane, or (c) canbe used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. Preferably, itbinds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In other embodiments, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells.

Exemplary vitamins include vitamin A, E, and K. Other exemplary vitaminsinclude are B vitamin, e.g., folic acid, B12, riboflavin, biotin,pyridoxal or other vitamins or nutrients taken up by target cells suchas liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:11). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:12) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:13) and theDrosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:14) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA further comprises a carbohydrate. The carbohydrate conjugated iRNAis advantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which can be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which can be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri-, and oligosaccharides containingfrom about 4, 5, 6, 7, 8, or 9 monosaccharide units), andpolysaccharides such as starches, glycogen, cellulose and polysaccharidegums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7,or C8) sugars; di- and trisaccharides include sugars having two or threemonosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In another embodiment,a carbohydrate conjugate for use in the compositions and methods of theinvention is selected from the group consisting of:

In one embodiment, the monosaccharide is an N-acetylgalactosamine, suchas

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Inanother embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the presentinvention include those described in PCT Publication Nos. WO 2014/179620and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, orsubstituted aliphatic. In one embodiment, the linker is about 1-24atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16,7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times, 20, times, 30times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or100 times faster in a target cell or under a first reference condition(which can, e.g., be selected to mimic or represent intracellularconditions) than in the blood of a subject, or under a second referencecondition (which can, e.g., be selected to mimic or represent conditionsfound in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential, or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 timesfaster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood or serum (or under invitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, and —O—P(S)(H)—S—. A preferred embodiment is—O—P(O)(OH)—O—. These candidates can be evaluated using methodsanalogous to those described above. iii. Acid cleavable linking groupsIn other embodiments, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or byagents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include, but are not limited to,esters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include but are not limited to

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to abivalent or trivalent branched linker selected from the group ofstructures shown in any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A),P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A),T^(5B), T^(5C) are each independently for each occurrence absent, CO,NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, or CH₂O; Q^(2A), Q^(2B), Q^(3A),Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently foreach occurrence absent, alkylene, substituted alkylene wherein one ormore methylenes can be interrupted or terminated by one or more of O, S,S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C, or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)OC(O)NH, NHCH(R^(a))C(O), —C(O)CH(R″)—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B), andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative US patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; and 8,106,022, the entire contents of each ofwhich are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAi agents, that containtwo or more chemically distinct regions, each made up of at least onemonomer unit, i.e., a nucleotide in the case of a dsRNA compound. TheseiRNAs typically contain at least one region wherein the RNA is modifiedso as to confer upon the iRNA increased resistance to nucleasedegradation, increased cellular uptake, or increased binding affinityfor the target nucleic acid. An additional region of the iRNA can serveas a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of iRNA inhibition of gene expression.Consequently, comparable results can often be obtained with shorteriRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof RNAs bearing an aminolinker at one or more positions of the sequence.The amino group is then reacted with the molecule being conjugated usingappropriate coupling or activating reagents. The conjugation reactioncan be performed either with the RNA still bound to the solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disease, disorder, or condition associated withXDH gene expression) can be achieved in a number of different ways. Forexample, delivery may be performed by contacting a cell with an iRNA ofthe invention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising an iRNA,e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may beperformed indirectly by administering one or more vectors that encodeand direct the expression of the iRNA. These alternatives are discussedfurther below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when a dsRNAi agent isadministered locally. For example, intraocular delivery of a VEGF dsRNAby intravitreal injection in cynomolgus monkeys (Tolentino, M J, et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA to thetarget tissue and avoid undesirable off-target effects. iRNA moleculescan be modified by chemical conjugation to lipophilic groups such ascholesterol to enhance cellular uptake and prevent degradation. Forexample, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O, et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H, et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R, et al(2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N, et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal,A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A.(2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu,S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, etal (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm.Res. 16:1799-1804). In some embodiments, an iRNA forms a complex withcyclodextrin for systemic administration. Methods for administration andpharmaceutical compositions of iRNAs and cyclodextrins can be found inU.S. Pat. No. 7,427,605, which is herein incorporated by reference inits entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the XDH gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A, et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of an XDH gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery, e.g., bysubcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. Thepharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an XDH gene.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an XDH gene. In general, asuitable dose of an iRNA of the invention will be in the range of about0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. Typically, a suitable dose of an iRNA ofthe invention will be in the range of about 0.1 mg/kg to about 5.0mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg. A repeat-doseregimine may include administration of a therapeutic amount of iRNA on aregular basis, such as every other day or once a year. In certainembodiments, the iRNA is administered about once per month to about onceper quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. For example, after administration weekly orbiweekly for three months, administration can be repeated once permonth, for six months, or a year; or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a composition can include a single treatment or aseries of treatments. Estimates of effective dosages and in vivohalf-lives for the individual iRNAs encompassed by the invention can bemade using conventional methodologies or on the basis of in vivo testingusing an appropriate animal model, as known in the art. For example,both genetic and induced models of hyperuricemia are known in the art.Genetic models of hyperuricemia include the B6; 129S7-Uox^(tm1Bay)/Jmouse available from Jackson Laboratory(/jaxmice.jax.org/strain/002223.html) which develops hyperuricemia, with10-fold higher levels of serum uric acid levels. Alternatively,hyperuricemia can be included in rats by feeing with a uricase inhibitor(oxonic acid) in the diet (Mazzali et al., Hypertension 38:1101-1106,2001; Habu et al., Biochem. Pharmacol. 66:1107-1114, 2003). Unlikehumans, rats and mice have uricase which metabolises uric acid,therefore, the enzyme must be inhibited in small animal models. Suchmodels and considerations are well known in the art.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal, or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricularadministration.

The iRNA can be delivered in a manner to target a particular tissue(e.g., vascular endothelial cells).

Pharmaceutical compositions and formulations for topical or transdermaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable. Coated condoms,gloves and the like can also be useful. Suitable topical formulationsinclude those in which the iRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). iRNAs featured in the invention can be encapsulatedwithin liposomes or can form complexes thereto, in particular tocationic liposomes. Alternatively, iRNAs can be complexed to lipids, inparticular to cationic lipids. Suitable fatty acids and esters includebut are not limited to arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA. The lipophilic material isolates the aqueous interiorfrom an aqueous exterior, which typically does not include the iRNAcomposition, although in some examples, it may. Liposomes are useful forthe transfer and delivery of active ingredients to the site of action.Because the liposomal membrane is structurally similar to biologicalmembranes, when liposomes are applied to a tissue, the liposomal bilayerfuses with bilayer of the cellular membranes. As the merging of theliposome and cell progresses, the internal aqueous contents that includethe iRNA are delivered into the cell where the iRNA can specificallybind to a target RNA and can mediate RNA interference. In some cases theliposomes are also specifically targeted, e.g., to direct the iRNA toparticular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natd. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natd. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of two or more of phospholipid, phosphatidylcholine, andcholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185 and 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M)I, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M)i, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In some embodiments, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg,Md.) is an effective agent for the delivery of highly anionic nucleicacids into living tissue culture cells that comprise positively chargedDOTMA liposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNAs can bedelivered, for example, subcutaneously by infection in order to deliveriRNAs to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inWO/2008/042973.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of iRNA, analkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds.Exemplary micelle forming compounds include lecithin, hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid, glycolic acid,lactic acid, chamomile extract, cucumber extract, oleic acid, linoleicacid, linolenic acid, monoolein, monooleates, monolaurates, borage oil,evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine andpharmaceutically acceptable salts thereof, glycerin, polyglycerin,lysine, polylysine, triolein, polyoxyethylene ethers and analoguesthereof, polidocanol alkyl ethers and analogues thereof,chenodeoxycholate, deoxycholate, and mixtures thereof. The micelleforming compounds may be added at the same time or after addition of thealkali metal alkyl sulphate. Mixed micelles will form with substantiallyany kind of mixing of the ingredients but vigorous mixing in order toprovide smaller size micelles.

In one method a first micellar composition is prepared which containsthe RNAi and at least the alkali metal alkyl sulphate. The firstmicellar composition is then mixed with at least three micelle formingcompounds to form a mixed micellar composition. In another method, themicellar composition is prepared by mixing the RNAi, the alkali metalalkyl sulphate and at least one of the micelle forming compounds,followed by addition of the remaining micelle forming compounds, withvigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol or m-cresol may be added with the micelle formingingredients. An isotonic agent such as glycerin may also be added afterformation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAi agents of in the invention may be fully encapsulatedin a lipid formulation, e.g., a LNP, or other nucleic acid-lipidparticle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; USPublication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech GI), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In some embodiments, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles.

In some embodiments, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle. In one embodiment, thelipidoid ND98-4HCl (MW 1487) (see U.S. patent application Ser. No.12/056,230, filed Mar. 26, 2008, which is incorporated herein byreference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (AvantiPolar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e.,LNP01 particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-dsRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMAdioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in International PatentApplication No. PCT/US2010/022614, filed on Jan. 29, 2010, the entirecontent of which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in US PatentPublication No. 2010/0324120, filed on Jun. 10, 2010, the entirecontents of which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., InternationalPatent Application Number PCT/US09/63933, filed on Nov. 10, 2009, theentire contents of which are hereby incorporated by reference.

C12-200 comprising formulations are described in PCT Publication No. WO2010/129709, the entire contents of which are hereby incorporated byreference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acids oresters or salts thereof, bile acids or salts thereof. Suitable bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG), and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses, and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents, and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds, and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids, and self-emulsifying semisolids. Formulationsinclude those that target the liver when treating hepatic disorders suchas hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The iRNAs of the present invention can be prepared and formulated asemulsions. Emulsions are typically heterogeneous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution either in the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate, and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andantioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, andparenteral routes, and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the iRNAs are formulated asmicroemulsions. A microemulsion can be defined as a system of water,oil, and amphiphile which is a single optically isotropic andthermodynamically stable liquid solution (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij® 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex® 300, Captex® 355,Capmul® MCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils, and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill® 3),Labrasol®, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsor aromatic substances and the like which do not deleteriously interactwith the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA and (b) one or more agents whichfunction by a non-iRNA mechanism and which are useful in treating anXDH-associated disorder.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity.

The dosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods featured in the invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby XDH expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VI. Methods For Inhibiting XDH Expression

The present invention also provides methods of inhibiting expression ofan XDH gene in a cell. The methods include contacting a cell with anRNAi agent, e.g., double stranded RNAi agent, in an amount effective toinhibit expression of XDH in the cell, thereby inhibiting expression ofXDH in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNAi agent,may be done in vitro or in vivo. Contacting a cell in vivo with the iRNAincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the iRNA. Combinations of in vitro and in vivomethods of contacting a cell are also possible. Contacting a cell may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In preferred embodiments, thetargeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, orany other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an XDH” is intended to refer toinhibition of expression of any XDH gene (such as, e.g., a mouse XDHgene, a rat XDH gene, a monkey XDH gene, or a human XDH gene) as well asvariants or mutants of an XDH gene. Thus, the XDH gene may be awild-type XDH gene, a mutant XDH gene (such as a mutant XDH gene givingrise to abnormal uric acid metabolism), or a transgenic XDH gene in thecontext of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of an XDH gene” includes any level of inhibitionof an XDH gene, e.g., at least partial suppression of the expression ofan XDH gene, inhibition of XDH in certain cells or tissues. Theexpression of the XDH gene may be assessed based on the level, or thechange in the level, of any variable associated with XDH geneexpression, e.g., XDH mRNA level or XDH protein level. This level may beassessed in an individual cell or in a group of cells, including, forexample, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with XDH expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of anXDH gene is inhibited by at least 15%, 20%, 25%, preferably at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or to below the level of detection of the assay. In some embodiments,the inhibition of expression of an XDH gene results in normalization ofthe level of the XDH gene such that the difference between the levelbefore treatment and a normal control level is reduced by at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Insome embodiments, the inhibition is a clinically relevant inhibition.

Inhibition of the expression of an XDH gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which an XDH gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an iRNA of theinvention, or by administering an iRNA of the invention to a subject inwhich the cells are or were present) such that the expression of an XDHgene is inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas not or have not been so treated (control cell(s) not treated with aniRNA or not treated with an iRNA targeted to the gene of interest). Inpreferred embodiments, the inhibition is assessed by the method providedin Example 2 with in vitro assays being performed in an appropriatelymatched cell line with the duplex at a 10 nM concentration, andexpressing the level of mRNA in treated cells as a percentage of thelevel of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of an XDH gene may beassessed in terms of a reduction of a parameter that is functionallylinked to XDH gene expression, e.g., serum uric acid levels. XDH genesilencing may be determined in any cell expressing XDH, eitherendogenous or heterologous from an expression construct, and by anyassay known in the art.

Inhibition of the expression of an XDH protein may be manifested by areduction in the level of the XDH protein that is expressed by a cell orgroup of cells (e.g., the level of protein expressed in a sample derivedfrom a subject). As explained above, for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells. For example,inhibition of expression can be inhibition of expression in liver cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of an XDH gene includes a cell or group ofcells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of XDH mRNA that is expressed by a cell or group of cells, orthe level of circulating XDH mRNA, may be determined using any methodknown in the art for assessing mRNA expression. In one embodiment, thelevel of expression of XDH in a sample is determined by detecting atranscribed polynucleotide, or portion thereof, e.g., mRNA of the XDHgene. RNA may be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits(Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formatsutilizing ribonucleic acid hybridization include nuclear run-on assays,RT-PCR, RNase protection assays, northern blotting, in situhybridization, and microarray analysis. In preferred embodiments, theRT-PCR assay provided in Example 2, with in vitro assays being performedin an appropriately matched cell line with the duplex at a 10 nMconcentration, is used to detect the level of expression of the mRNA.

In some embodiments, the level of expression of XDH is determined usinga nucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific XDH.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to XDHmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of XDH mRNA.

An alternative method for determining the level of expression of XDH ina sample involves the process of nucleic acid amplification or reversetranscriptase (to prepare cDNA) of for example mRNA in the sample, e.g.,by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S.Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl.Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988)Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniqueswell known to those of skill in the art. These detection schemes areespecially useful for the detection of nucleic acid molecules if suchmolecules are present in very low numbers. In particular aspects of theinvention, the level of expression of XDH is determined by quantitativefluorogenic RT-PCR (i.e., the TaqMan™ System) using the methods providedherein.

The expression levels of XDH mRNA may be monitored using a membrane blot(such as used in hybridization analysis such as northern, Southern, dot,and the like), or microwells, sample tubes, gels, beads or fibers (orany solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of XDH expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of XDH protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention in thetreatment of an XDH-related disease is assessed by a decrease in XDHmRNA level (by liver biopsy) or XDH protein level, typically determinedin serum.

In some embodiments, the efficacy of the compositions methods of theinvention in the treatment of hyperuricemia is demonstrated by asignificant decrease in chronic uric acid levels in a subject to 6.8mg/dl or less (the level of solubility of uric acid in serum),preferably 6 mg/dl or less. Studies of subjects with inherited disordersassociated with profound, lifelong hypouricemia indicate thatmaintaining serum uric acid near or below 2 mg/dl is safe (Hershfeld,Curr. Opin. Rheumatol. 21:138-142, 2009). As elevated levels of serumuric acid are associated with a number of diseases and conditions, adecrease in chronic uric acid levels towards, or preferably to, normallevels, would treat those conditions. The compositions and methods ofthe invention can also be used to treat aymptomatic hyperuricemia.Normalizing hyperuricemia prevents one or more of the comorbiditiesassociated with hyperuricemia including, but not limited to, gout,NAFLD, NASH, metabolic disorder, insulin resistance, cardiovasculardisease, hypertension, type 2 diabetes, and conditions linked tooxidative stress e.g., chronic low grade inflammation; or otherXDH-associated disease. In some embodiments of the methods of theinvention, the iRNA is administered to a subject such that the iRNA isdelivered to a specific site within the subject. The inhibition ofexpression of XDH may be assessed using measurements of the level orchange in the level of XDH mRNA or XDH protein in a sample derived froma specific site within the subject, e.g., the liver.

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

VI. Methods of Treating or Preventing XDH-Associated Diseases

The present invention provides therapeutic and prophylactic methodswhich include administering to a subject having an XDH-associateddisease, disorder, and/or condition, or prone to developing, anXDH-associated disease, disorder, and/or condition, compositionscomprising an iRNA agent targeting an XDH gene. Non-limiting examples ofXDH-associated diseases include, for example, gout, NAFLD, NASH,metabolic disorder, insulin resistance, cardiovascular disease,hypertension, type 2 diabetes, and conditions linked to oxidative stresse.g., chronic low grade inflammation.

The methods of the invention are useful for treating a subject having anXDH-associated disease, e.g., a subject that would benefit fromreduction in XDH gene expression and/or XDH protein production.

In one aspect, the invention provides methods of preventing at least onesign or symptom in a subject susceptible to or having an XDH-associateddisease, e.g., gout, NAFLD, NASH, metabolic disorder, insulinresistance, cardiovascular disease, hypertension, type 2 diabetes, andconditions linked to oxidative stress e.g., chronic low gradeinflammation. The methods include administering to the subject aprohylactically effective amount of the iRNA agent, e.g. dsRNA,pharmaceutical compositions, or vectors of the invention, therebypreventing at least one sign or symptom in a subject having anXDH-associated disease.

In one embodiment, an iRNA agent targeting XDH is administered to asubject having an XDH-associated disease such that the expression of anXDH gene, e.g., in a cell, tissue, blood or other tissue or fluid of thesubject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% or more, or to a level below the level of detection of theassay, when the dsRNA agent is administered to the subject.

The methods and uses of the invention include administering acomposition described herein such that expression of the target XDH geneis decreased for an extended duration, e.g., at least one month,preferably at least three months.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with anXDH-associated disease. By “reduction” in this context is meant astatistically or clinically significant decrease in such level. Thereduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Comparisonof the later readings with the initial readings, or historicallyrelevant population controls, provide a physician an indication ofwhether the treatment is effective. It is well within the ability of oneskilled in the art to monitor efficacy of treatment or prevention bymeasuring any one of such parameters, or any combination of parameters.In connection with the administration of an iRNA targeting an XDH geneor pharmaceutical composition thereof, “effective against” anXDH-associated disease indicates that administration in a clinicallyappropriate manner results in a beneficial effect for at least astatistically significant fraction of patients, such as improvement ofsymptoms, a cure, a reduction in disease, extension of life, improvementin quality of life, or other effect generally recognized as positive bymedical doctors familiar with treating an XDH-associated disease and therelated causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

XDH expression may be inhibited, e.g., in a liver cell of a subject, byat least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or95, or to a level below the level of detection of the assay. The methodsof the invention do not require knockdown of expression in all celltypes in which XDH is expressed.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the XDH gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection. In certain embodiments, the compositions areadministered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof XDH, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an XDH gene in a mammal. The methodsinclude administering to the mammal a composition comprising an iRNAthat targets an XDH gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the XDH gene, thereby inhibiting expression of the XDHgene in the cell. Reduction in gene expression can be assessed by anymethods known it the art and by methods, e.g. qRT-PCR, described herein.Reduction in protein production can be assessed by any methods known itthe art and by methods, e.g. ELISA, described herein. In one embodiment,a puncture liver biopsy sample serves as the tissue material formonitoring the reduction in the XDH gene or protein expression.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction or inhibition of XDH expression, ina therapeutically effective amount of an iRNA targeting an XDH gene or apharmaceutical composition comprising an iRNA targeting an XDH gene.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of XDH geneexpression are those having hyperuricemia as demonstrated by a chronicuric acid level of at least 6.8 mg/dl. It is expected that normalizinghyperuricemia would prevent one or more of the comorbidities associatedwith hyperuricemia including, but not limited to, gout, NAFLD, NASH,metabolic disorder, insulin resistance, cardiovascular disease,hypertension, type 2 diabetes, and conditions linked to oxidative stresse.g., chronic low grade inflammation; or other XDH-associated disease.

A. Hyperuricemia

Serum uric acid levels are not routinely obtained as clinical labvalues. However, hyperuricemia is associated with a number of diseasesand conditions including, gout, NAFLD, NASH, metabolic disorder, insulinresistance, cardiovascular disease, hypertension, type 2 diabetes, andconditions linked to oxidative stress e.g., chronic low gradeinflammation. Normalization of uric acid levels is useful in theprevention or treatment of one or more conditions associated withelevated serum uric acid levels. Further, a subject derives clinicalbenefit from normalization of serum uric acid levels towards or to anormal serum uric acid level, e.g., no more than 6.8 mg/dl, preferablyno more than 6 mg/dl, even in the absence of overt signs or symptoms ofone or more conditions associated with elevated uric acid, e.g., gout,NAFLD, NASH, metabolic disorder, insulin resistance, cardiovasculardisease, hypertension, type 2 diabetes, or conditions linked tooxidative stress e.g., chronic low grade inflammation. Methods to detectand monitor uric acid in serum or other subject samples are known in theart. Uric acid levels can be detected, for example usingcarbonate-phosphotungstate method, spectrophotometric uricase method, orchromatography methods such as HPLC or LCMS.

Allopurinol is a xanthine oxidase inhibitor that is used to reduce serumuric acid levels for the treatment of a number of conditions, e.g.,gout, cardiovascular disease including ischemia-reperfusion injury,hypertension, atherosclerosis, and stroke, and inflammatory diseases(Pacher et al., Pharma. Rev. 58:87-114, 2006). However, the use ofallopurinol is contraindicated in subjects with impaired renal function,e.g., chronic kidney disease, hypothyroidism, hyperinsulinemia, orinsulin resistance; or in subjects predisposed to kidney disease orimpaired renal function, e.g., subjects with hypertension, metabolicdisorder, diabetes, and the elderly. Further, allopurinol should not betaken by subjects taking oral coagulants or probencid as well assubjects taking diuretics, especially thiazide diuretics or other drugsthat can reduce kidney function or have potential kidney toxicity.

In certain embodiments, the compositions and methods of the inventionare for use in treatment of subjects with hyperuricemia and impairedrenal function. For example, in certain embodiments, the compositionsand methods of the invention are for use in subjects with hyperuricemiaand chronic kidney disease. In certain embodiments, the compositions andmethods are for use in subjects with hyperuricemia who are sufferingfrom one or more of cardiovascular disease, metabolic disorder, insulinresistance, hyperinsulinemia, diabetes, hypothyroidism, or inflammatorydisease; or elderly subjects (e.g., over 65). In certain embodiments,the compositions and methods are for use in subjects with hyperuricemiawho are also taking a drug that can reduce kidney function asdemonstrated by the drug label. For example, in certain embodiments thecompositions and methods of the invention are for use in subjects withhyperuricemia who are being treated with oral coagulants or probencid.For example, in certain embodiments the compositions and methods of theinvention are for use in subjects with hyperuricemia who are beingtreated with diuretics, especially thiazide diuretics.

In certain embodiments, the compositions and methods of the inventionare used in combination with other compositions and methods to treathyperuricemia, e.g., allopurinol, oxypurinol, febuxostat.

B. Gout

Gout affects approximately 1 in 40 adults, most commonly men between30-60 years of age. Gout less commonly affects women. Gout is one of afew types of arthritis where future damage to joints can be avoided bytreatment. Gout is characterized by recurrent attacks of acuteinflammatory arthritis caused by an inflammatory reaction to uric acidcrystals in the joint due to hyperuricemia resulting from insufficientrenal clearance of uric acid or excessive uric acid production. Fructoseassociated gout is associated with variants of transporters expressed inthe kidney, intestine, and liver. Gout is characterized by the formationand deposition of tophi, monosodium urate (MSU) crystals, in the jointsand subcutaneously. Pain associated with gout is not related to the sizeof the tophi, but is a result of an immune response against the MSUcrystals. There is a linear inverse relation between serum uric acid andthe rate of decrease in tophus size. For example, in one study of 18patients with non-tophaceous gout, serum uric acid declined to 2.7-5.4mg/dL (0.16-0.32 mM) in all subjects within 3 months of starting uratelowering therapy (Pascual and Sivera, Ann. Rheum. Dis. 66:1056-1058).However, it took 12 months with normalized serum uric acid for MSUcrystals to disappear from asymptomatic knee or first MTP joints inpatients who had gout for less than 10 years, vs. 18 months in thosewith gout for more than 10 years. Therefore, effective treatment of goutdoes not require complete clearance of tophi or resolution of allsymptoms, e.g., joint pain and swelling, inflammation, but simply areduction in at least one sign or symptom of gout, e.g., reduction inseverity or frequency of gout attacks, in conjunction with a reductionin serum urate levels.

Currently available treatments for gout are contraindicated orineffective in a number of subjects. Allopurinol, a common first linetreatment to reduce uric acid levels in subjects with gout, iscontraindicated in a number of populations, especially those withcompromised renal function, as discussed above. Further, a number ofsubjects fail treatment with allopurinol, e.g., subjects who suffer goutflares despite treatment, or subjects who suffer from rashes orhypersensitivity reactions associated with allopurinol.

In certain embodiments, the compositions and methods of the inventionare for use in treatment of subjects with gout and impaired renalfunction. For example, in certain embodiments, the compositions andmethods of the invention are for use in subjects with gout and chronickidney disease. In certain embodiments, the compositions and methods arefor use in subjects with gout who are suffering from one or more ofcardiovascular disease, metabolic disorder, insulin resistance,hyperinsulinemia, diabetes, hypothyroidism, or inflammatory disease; orelderly subjects (e.g., over 65). In certain embodiments, thecompositions and methods are for use in subjects with gout who are alsotaking a drug that can reduce kidney function as demonstrated by thedrug label. For example, in certain embodiments the compositions andmethods of the invention are for use in subjects with gout who are beingtreated with oral coagulants or probencid. For example, in certainembodiments the compositions and methods of the invention are for use insubjects with gout who are being treated with diuretics, especiallythiazide diuretics. For example, in certain embodiments the compositionsand methods of the invention are for use in subjects with gout who havefailed treatment with allopurinol.

In certain embodiments, the compositions and methods of the inventionare used in combination with other agents to reduce serum uric acid. Incertain embodiments, the compositions and methods of the invention areused in combination with agents for treatment of symptoms of gout, e.g.,analgesic or anti-inflammatory agents, e.g., NSAIDS.

C. NAFLD

NAFLD is associated with hyperuricemia (Xu et al., J. Hepatol.62:1412-1419, 2015). The definition of nonalcoholic fatty liver disease(NAFLD) requires that (a) there is evidence of hepatic steatosis, eitherby imaging or by histology and (b) there are no causes for secondaryhepatic fat accumulation such as significant alcohol consumption, use ofsteatogenic medication or hereditary disorders. In the majority ofpatients, NAFLD is associated with metabolic risk factors such asobesity, diabetes mellitus, and dyslipidemia. NAFLD is histologicallyfurther categorized into nonalcoholic fatty liver (NAFL) andnonalcoholic steatohepatitis (NASH). NAFL is defined as the presence ofhepatic steatosis with no evidence of hepatocellular injury in the formof ballooning of the hepatocytes. NASH is defined as the presence ofhepatic steatosis and inflammation with hepatocyte injury (ballooning)with or without fibrosis (Chalasani et al., Hepatol. 55:2005-2023,2012). It is generally agreed that patients with simple steatosis havevery slow, if any, histological progression, while patients with NASHcan exhibit histological progression to cirrhotic-stage disease. Thelong term outcomes of patients with NAFLD and NASH have been reported inseveral studies. Their findings can be summarized as follows; (a)patients with NAFLD have increased overall mortality compared to matchedcontrol populations, (b) the most common cause of death in patients withNAFLD, NAFL, and NASH is cardiovascular disease, and (c) patients withNASH (but not NAFL) have an increased liver-related mortality rate. In amouse model of NAFLD, treatment with allopurinol both prevented thedevelopment of hepatic steatosis, but also significantly amelioratedestablished hepatic steatosis in mice (Xu et al., J. Hepatol.62:1412-1419, 2015).

As discussed above, treatment with allopurinol is contraindicated in anumber of populations, especially those with compromised renal function.

In certain embodiments, the compositions and methods of the inventionare for use in treatment of subjects with NAFLD and impaired renalfunction. For example, in certain embodiments, the compositions andmethods of the invention are for use in subjects with NAFLD and chronickidney disease. In certain embodiments, the compositions and methods arefor use in subjects with NAFLD who are suffering from one or more ofcardiovascular disease, metabolic disorder, insulin resistance,hyperinsulinemia, diabetes, hypothyroidism, or inflammatory disease; orelderly subjects (e.g., over 65). In certain embodiments, thecompositions and methods are for use in subjects with NAFLD who are alsotaking a drug that can reduce kidney function as demonstrated by thedrug label. For example, in certain embodiments the compositions andmethods of the invention are for use in subjects with NAFLD who arebeing treated with oral coagulants or probencid. For example, in certainembodiments the compositions and methods of the invention are for use insubjects with NAFLD who are being treated with diuretics, especiallythiazide diuretics. For example, in certain embodiments the compositionsand methods of the invention are for use in subjects with NAFLD who havefailed treatment with allopurinol.

In certain embodiments, the compositions and methods of the inventionare used in combination with other agents to reduce serum uric acid. Incertain embodiments, the compositions and methods of the invention areused in combination with agents for treatment of symptoms of NAFLD.

D. Cardiovascular Disease

Cardiovascular disease has been associated with hyperuricemia.Allopurinol has been demonstrated to be effective in the treatment ofcardiovascular disease in animal models and humans including myocardialinfarction, ischemia-reperfusion injury, hypoxia, ischemic heartdisease, heart failure, hypercholesterolemia, and hypertension (Pacheret al., Pharma. Rev. 58:87-114, 2006). As discussed above, treatmentwith allopurinol is contraindicated in a number of populations,especially those with compromised renal function.

In certain embodiments, the compositions and methods of the inventionare for use in treatment of subjects with cardiovascular disease andimpaired renal function. For example, in certain embodiments, thecompositions and methods of the invention are for use in subjects withcardiovascular disease and chronic kidney disease. In certainembodiments, the compositions and methods are for use in subjects withcardiovascular disease who are suffering from one or more of metabolicdisorder, insulin resistance, hyperinsulinemia, diabetes,hypothyroidism, or inflammatory disease. In certain embodiments, thecompositions and methods are for use in subjects with cardiovasculardisease who are also taking a drug that can reduce kidney function asdemonstrated by the drug label. For example, in certain embodiments thecompositions and methods of the invention are for use in subjects withcardiovascular disease who are being treated with oral coagulants orprobencid. For example, in certain embodiments the compositions andmethods of the invention are for use in subjects with cardiovasculardisease who are being treated with diuretics, especially thiazidediuretics. For example, in certain embodiments the compositions andmethods of the invention are for use in subjects with cardiovasculardisease who have failed treatment with allopurinol.

In certain embodiments, the compositions and methods of the inventionare used in combination with other agents to reduce serum uric acid. Incertain embodiments, the compositions and methods of the invention areused in combination with agents for treatment of symptoms ofcardiovascular disease, e.g., agents to decrease blood pressure, e.g.,diuretics, beta-blockers, ACE inhibitors, angiotensin II receptorblockers, calcium channel blockers, alpha blockers, alpha-2 receptorantagonists, combined alpha- and beta-blockers, central agonists,peripheral adrenergic inhibitors, and blood vessel dialators; or agentsto decrease cholesterol, e.g., statins, selective cholesterol absorptioninhibitors, resins, or lipid lowering therapies.

E. Metabolic Syndrome, Insulin Resistance, and Type 2 Diabetes

Metabolic syndrome, insulin resistance, and type 2 diabetes areassociated with hyperuricemia (Cardoso et al., J. Pediatr. 89:412-418,2013).

Metabolic syndrome is characterized by a cluster of conditions definedas at least three of the five following metabolic risk factors:

-   -   1. Large waistline (≥35 inches for women or ≥40 inches for men);    -   2. High triglyceride level (≥150 mg/dl);    -   3. Low HDL cholesterol (≤50 mg/dl for women or ≤40 mg/dl for        men);    -   4. Elevated blood pressure (≥130/85) or on medicine to treat        high blood pressure; and    -   5. High fasting blood sugar (≥100 mg/dl) or being in medicine to        treat high blood sugar.

Insulin resistance is characterized by the presence of at least one of:

-   -   1. A fasting blood glucose level of 100-125 mg/dL taken at two        different times; or    -   2. An oral glucose tolerance test with a result of a glucose        level of 140-199 mg/dL at    -   2 hours after glucose consumption.

Type 2 diabetes is characterized by at least one of:

-   -   1. A fasting blood glucose level ≥126 mg/dL taken at two        different times;    -   2. A hemoglobin A1c (A1C) test with a result of ≥6.5% or higher;        or    -   3. An oral glucose tolerance test with a result of a glucose        level ≥200 mg/dL at 2 hours after glucose consumption.

Metabolic syndrome, insulin resistance, and type 2 diabetes are oftenassociated with decreased renal function or the potential for decreasedrenal function.

In certain embodiments, the compositions and methods of the inventionare for use in treatment of subjects with metabolic syndrome, insulinresistance, or type 2 diabetes and impaired renal function. For example,in certain embodiments, the compositions and methods of the inventionare for use in subjects with metabolic syndrome, insulin resistance, ortype 2 diabetes and chronic kidney disease. In certain embodiments, thecompositions and methods are for use in subjects with metabolicsyndrome, insulin resistance, or type 2 diabetes who are suffering fromone or more of cardiovascular disease, hypothyroidism, or inflammatorydisease; or elderly subjects (e.g., over 65). In certain embodiments,the compositions and methods are for use in subjects with metabolicsyndrome, insulin resistance, or type 2 diabetes who are also taking adrug that can reduce kidney function as demonstrated by the drug label.For example, in certain embodiments the compositions and methods of theinvention are for use in subjects with metabolic syndrome, insulinresistance, or type 2 diabetes who are being treated with oralcoagulants or probencid. For example, in certain embodiments thecompositions and methods of the invention are for use in subjects withmetabolic syndrome, insulin resistance, or type 2 diabetes who are beingtreated with diuretics, especially thiazide diuretics.

In certain embodiments, the compositions and methods of the inventionare used in combination with other agents to reduce serum uric acid. Incertain embodiments, the compositions and methods of the invention areused in combination with agents for treatment of symptoms of metabolicsyndrome, insulin resistance, or type 2 diabetes. In certainembodiments, subjects are treated with e.g., agents to decrease bloodpressure, e.g., diuretics, beta-blockers, ACE inhibitors, angiotensin IIreceptor blockers, calcium channel blockers, alpha blockers, alpha-2receptor antagonists, combined alpha- and beta-blockers, centralagonists, peripheral adrenergic inhibitors, and blood vessel dialators;agents to decrease cholesterol, e.g., statins, selective cholesterolabsorption inhibitors, resins, or lipid lowering therapies; or agents tonormalize blood sugar, e.g., metformin, sulfonylureas, meglitinides,thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor antagonists, andSGLT2 inhibitors.

The iRNA and additional therapeutic agents may be administered at thesame time or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times or by another method known in the artor described herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target XDH gene is decreasedin at least one tissue, e.g., in liver, such as for about 1, 2, 3, 4, 5,6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In oneembodiment, expression of the target XDH gene is decreased for anextended duration, e.g., at least about two, three, four days or more,e.g., about one week, two weeks, three weeks, or four weeks or longer,in at least one tissue, e.g., liver.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the target XDHgene. Compositions and methods for inhibiting the expression of thesegenes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention mayresult in a reduction of the severity, signs, symptoms, or markers ofsuch diseases or disorders in a patient with a disorder of elevatedserum uric acid. By “reduction” in this context is meant a statisticallysignificant decrease in such level. The reduction can be, for example,at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95%, or to below the level of detection of theassay used. For certain measures, a reduction of a marker to the lowestpossible level may not be desirable. For example, as used herein,reduction includes a lowering towards or to a normal serum uric acidlevel in the subject. For example, reduction includes lowering towardsor preferably to 6.8 mg/dl or less of serum uric acid. In certainembodiments, reduction includes lowering to 6 mg/dl or less of serumuric acid. Typically, a serum uric acid level of at least 2 mg/dl ispreferred. Similarly, triglyceride levels are preferably lowered (i.e.,normalized) to less than 150 mg/dl and fasting blood sugar is lowered(i.e., normalized) to less than 100 mg/dl. Such considerations are wellunderstood by those of skill in the art.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker, orany other measurable parameter appropriate for a given disease beingtreated or targeted for prevention. It is well within the ability of oneskilled in the art to monitor efficacy of treatment or prevention bymeasuring any one of such parameters, or any combination of parameters.For example, efficacy of treatment of an XDH related disorder.Diagnostic criteria for a number of XDH related disorders are providedabove. The exact criteria for treatment or prevention will depend on thedisease or condition present in the subject or the disease or conditionto which the subject is susceptible. Treatment and prevention typicallyinclude normalization or maintenance of normal clinical laboratoryvalues.

Comparisons of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of aniRNA targeting XDH or pharmaceutical composition thereof, “effectiveagainst” an XDH related disorder indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such as aimprovement of symptoms, a cure, a reduction in disease, extension oflife, improvement in quality of life, or other effect generallyrecognized as positive by medical doctors familiar with treatingXDH-related disorders.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment, e.g., normalization of a clinical laboratory value. Efficacyfor a given iRNA drug or formulation of that drug can also be judgedusing an experimental animal model for the given disease as known in theart. When using an experimental animal model, efficacy of treatment isevidenced when a statistically significant reduction in a marker orsymptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale. Anypositive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period oftime, on a regular basis. In certain embodiments, after an initialtreatment regimen, the treatments can be administered on a less frequentbasis. Administration of the iRNA can reduce XDH levels, e.g., in atleast one of a cell, tissue, blood, urine, or other compartment of thepatient by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection ofthe assay method used.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired daily dose of iRNA to a subject. The injections may berepeated over a period of time. The administration may be repeated on aregular basis. In certain embodiments, after an initial treatmentregimen, the treatments can be administered on a less frequent basis. Arepeat-dose regimen may include administration of a therapeutic amountof iRNA on a regular basis, such as every other day or to once a year.In certain embodiments, the iRNA is administered about once per month toabout once per quarter (i.e., about once every three months).

IX. Cell Type XDH Expression

XDH is expressed predominantly in the liver and intestine, However,adipocytes and other extrahepatic tissues express XDH and thus cancontribute significant amounts of uric acid in the body. As a result,silencing XDH mRNA in obese patients with high BMI may result in lessuric acid lowering than in lean patients. Additionally, silencing XDHmRNA in the liver may be insufficient to lower urate levels givenextrahepatic XDH expression. In patients with reduced, but not complete,urinary uric acid excretion, silencing liver XDH mRNA may beinsufficient to reduce urate burden from poor renal clearance (defectssuch as SLC2A9, ABCG2 etc.) Therefore, the efficacy of the iRNAcompounds of the invention to prevent or treat an XDH-associatedcondition, and the amount required for an effective dose, may depend, atleast in part, on the amount of fat present in the subject rather thanthe overall size of the subject. Therefore, in certain embodiments, thecompositions and methods of the invention are not for treatment or notfor treatment as a single agent in subjects who are obese, i.e., a bodymass index (BMI)≥30.0 wherein BMI is a person's weight in kilogramsdivided by the square of height in meters. A high BMI can be anindicator of high body fatness.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Sequence Listing, are herebyincorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA Design

A set of siRNAs targeting the human XDH “xanthine dehydrogenase” gene(human: NCBI refseqID NM_000379; NCBI GeneID: 7498), as well astoxicology-species XDH orthologs (cynomolgus monkey: XM_005576184;mouse: NM_011723; rat, NM_017154) were designed using custom R andPython scripts. The human NM_000379 REFSEQ mRNA, version 3, has a lengthof 5717 bases. The rationale and method for the set of siRNA designs isas follows: the predicted efficacy for every potential 19mer siRNA fromposition 80 through position 5717 (the coding region and 3′ UTR) wasdetermined with a linear model derived the direct measure of mRNAknockdown from more than 20,000 distinct siRNA designs targeting a largenumber of vertebrate genes. Subsets of the XDH siRNAs were designed withperfect or near-perfect matches between human, cynomolgus and rhesusmonkey. A further subset was designed with perfect or near-perfectmatches to mouse and rat XDH orthologs. For each strand of the siRNA, acustom Python script was used in a brute force search to measure thenumber and positions of mismatches between the siRNA and all potentialalignments in the target species transcriptome. Extra weight was givento mismatches in the seed region, defined here as positions 2-9 of theantisense oligonucleotide, as well the cleavage site of the siRNA,defined here as positions 10-11 of the antisense oligonucleotide. Therelative weight of the mismatches was 2.8; 1.2:1 for seed mismatches,cleavage site, and other positions up through antisense position 19.Mismatches in the first position were ignored. A specificity score wascalculated for each strand by summing the value of each weightedmismatch. Preference was given to siRNAs whose antisense score in humanand cynomolgus monkey was >=2.0 and predicted efficacy was >=50%knockdown of the XDH transcript.

siRNA Synthesis

XDH siRNA sequences are synthesized at 1 umol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support is controlled pore glass(500° A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites are obtained from Thermo-Fisher (Milwaukee, Wis.)and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modifiednucleosides are introduced in the sequences using the correspondingphosphoramidites. Synthesis of 3′ GalNAc conjugated single strands isperformed on a GalNAc modified CPG support. Custom CPG universal solidsupport is used for the synthesis of antisense single strands. Couplingtime for all phosphoramidites (100 mM in acetonitrile) is 5 minemploying 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M inacetonitrile). Phosphorothioate linkages are generated using a 50 mMsolution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass.,USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time is 3minutes. All sequences are synthesized with final removal of the DMTgroup (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides arecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagent at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection is performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 uL ofdimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent is added and thesolution was incubated for additional 20 min at 60° C. At the end ofcleavage and deprotection step, the synthesis plate is allowed to cometo room temperature and is precipitated by addition of 1 mL ofacetontile:ethanol mixture (9:1). The plates are cooled at −80° C. for 2hrs and the superanatant decanted carefully with the aid of a multichannel pipette. The oligonucleotide pellet is re-suspended in 20 mMNaOAc buffer and desalted using a 5 mL HiTrap size exclusion column (GEHealthcare) on an AKTA Purifier System equipped with an A905 autosamplerand a Frac 950 fraction collector. Desalted samples are collected in 96well plates. Samples from each sequence are analyzed by LC-MS to confirmthe identity, UV (260 nm) for quantification and a selected set ofsamples by IEX chromatography to determine purity.

Annealing of XDH single strands is performed on a Tecan liquid handlingrobot. Equimolar mixture of sense and antisense single strands arecombined and annealed in 96 well plates. After combining thecomplementary single strands, the 96 well plate is sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex is normalized to 10 uM in 1×PBS and then submitted for in vitroscreening assays.

A detailed list of the unmodified XDH sense and antisense strandsequences is shown in Table 3 and a detailed list of the modified XDHsense and antisense strand sequences is shown in Table 4.

Example 2—In Vitro Screening—Primary Mouse Hepatocytes and Primary CynoHepatocytes Cell Culture and Transfections:

Primary Mouse Hepatocytes (PMH) (GIBCO) and Primary Cyno Hepatocytes(PCH) (Celsis) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μlof Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. Forty l of William's EMedium (Life Tech) containing about 5×10³ cells were then added to thesiRNA mixture. Cells were incubated for 24 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl ofLysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand the supernatant was removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 hours at 37° C. Plates arethen incubated at 81° C. for 8 minutes.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μl of GAPDHTaqMan Probe (Hs99999905 ml), 0.5 μl XDH probe and 5 μl Lightcycler 480probe master mix (Roche Cat #04887301001) per well in a 384 well plates(Roche cat #04887301001). Real time PCR was performed in aLightCycler480 Real Time PCR system (Roche). Each duplex was assayed atleast two times and data were normalized to cells transfected with anon-targeting control siRNA.

To calculate relative fold change, real time data was analyzed using theΔΔCt method and normalized to assays performed with cells transfectedwith 10 nM AD-1955, a non-targeting control siRNA.

The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 17) cuuAcGcuGAGuAcuucGAdTsdT; ANTISENSE:(SEQ ID NO: 18) UCGAAGuACUcAGCGuAAGdTsdT.

Table 5 shows the results of a single dose screen in Primary CynomolgusHepatocytes and Primary Mouse Hepatocytes transfected with the indicatedmodified iRNAs.

Table 2. Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds.

Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Af2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3 dT 2′-deoxythymidine-3′-phosphate dC2′-deoxycytidine-3′-phosphate Y44 inverted abasic DNA(2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Tgn) Thymidine-glycolnucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphate (Aam)2′-O-(N-methylacetamide)adenosine-3′-phosphate

TABLE 3 Unmodified Sense and Antisense Strand Sequences of XDH dsRNAsPosition Anti- Position Sense SEQ in sense SEQ in Duplex OligoSense Sequence ID NM_ Oligo Antisense Sequence ID NM_ Name Name(5′ to 3′) NO 000379.3 Name (5′ to 3′) NO 000379.3 AD-70003 A-140499AACUGUGGAAGGAAUAGGAAA  19  339- A-140500 UUUCCUAUUCCUUCCACAGUUGU 105 337- 358_G21A 358_G21A AD-70049 A-140591 AACUGUGGAAGGAAUAGGAAA  20 339- A-140592 UUUCCUAUUCCUUCCACAGUUGU 106  337- 358_G21A 358_G21AAD-69981 A-140455 GAGGAGAUUGAGAAUGCCUUA  21  490- A-140456UAAGGCAUUCUCAAUCUCCUCCA 107  488- 509_C21A 509_C21A AD-70027 A-140547GAGGAGAUUGAGAAUGCCUUA  22  490- A-140548 UAAGGCAUUCUCAAUCUCCUCCA 108 488- 509_C21A 509_C21A AD-69970 A-140433 GAGAUUGAGAAUGCCUUCCAA  23 493-512 A-140434 UUGGAAGGCAUUCUCAAUCUCCU 109  491-512 AD-70016 A-140525GAGAUUGAGAAUGCCUUCCAA  24  493-512 A-140526 UUGGAAGGCAUUCUCAAUCUCCU 110 491-512 AD-70007 A-140507 GUUCAAGAAUAUGCUGUUUCA  25  888- A-140508UGAAACAGCAUAUUCUUGAACUU 111  886- 907_C21A 907_C21A AD-70053 A-140599GUUCAAGAAUAUGCUGUUUCA  26  888- A-140600 UGAAACAGCAUAUUCUUGAACUU 112 886- 907_C21A 907_C21A AD-69995 A-140483 AAAAGACAGAGGUGUUCAGAA  271046- A-140484 UUCUGAACACCUCUGUCUUUUGG 113 1044- 1065_G21A 1065_G21AAD-70041 A-140575 AAAAGACAGAGGUGUUCAGAA  28 1046- A-140576UUCUGAACACCUCUGUCUUUUGG 114 1044- 1065_G21A 1065_G21A AD-69973 A-140439AAAGACAGAGGUGUUCAGAGA  29 1047- A-140440 UCUCUGAACACCUCUGUCUUUUG 1151045- 1066_G21A 1066_G21A AD-70019 A-140531 AAAGACAGAGGUGUUCAGAGA  301047- A-140532 UCUCUGAACACCUCUGUCUUUUG 116 1045- 1066_G21A 1066_G21AAD-69975 A-140443 CGGAGAGAAGAUGACAUUGCA  31 1357- A-140444UGCAAUGUCAUCUUCUCUCCGGG 117 1355- 1376_C21A 1376_C21A AD-70021 A-140535CGGAGAGAAGAUGACAUUGCA  32 1357- A-140536 UGCAAUGUCAUCUUCUCUCCGGG 1181355- 1376_C21A 1376_C21A AD-69983 A-140459 AGAGAAGAUGACAUUGCCAAA  331360- A-140460 UUUGGCAAUGUCAUCUUCUCUCC 119 1358- 1379_G21A 1379_G21AAD-70029 A-140551 AGAGAAGAUGACAUUGCCAAA  34 1360- A-140552UUUGGCAAUGUCAUCUUCUCUCC 120 1358- 1379_G21A 1379_G21A AD-69986 A-140465GAAGAUGACAUUGCCAAGGUA  35 1363-1382 A-140466 UACCUUGGCAAUGUCAUCUUCUC 1211361-1382 AD-70032 A-140557 GAAGAUGACAUUGCCAAGGUA  36 1363-1382 A-140558UACCUUGGCAAUGUCAUCUUCUC 122 1361-1382 AD-69976 A-140445CAGUCUGAGGAGGACAUGGUA  37 1783- A-140446 UACCAUGUCCUCCUCAGACUGAC 1231781- 1802_G21A 1802_G21A AD-70022 A-140537 CAGUCUGAGGAGGACAUGGUA  381783- A-140538 UACCAUGUCCUCCUCAGACUGAC 124 1781- 1802_G21A 1802_G21AAD-69987 A-140467 AAGGAUAAGGUUACUUGUGUU  39 2053-2072 A-140468AACACAAGUAACCUUAUCCUUCG 125 2051-2072 AD-70033 A-140559AAGGAUAAGGUUACUUGUGUU  40 2053-2072 A-140560 AACACAAGUAACCUUAUCCUUCG 1262051-2072 AD-69990 A-140473 AGGAUAAGGUUACUUGUGUUA  41 2054- A-140474UAACACAAGUAACCUUAUCCUUC 127 2052- 2073_G21A 2073_G21A AD-70036 A-140565AGGAUAAGGUUACUUGUGUUA  42 2054- A-140566 UAACACAAGUAACCUUAUCCUUC 1282052- 2073_G21A 2073_G21A AD-69989 A-140471 CAUAUCAUUGGUGCUGUGGUU  432077-2096 A-140472 AACCACAGCACCAAUGAUAUGCC 129 2075-2096 AD-70035A-140563 CAUAUCAUUGGUGCUGUGGUU  44 2077-2096 A-140564AACCACAGCACCAAUGAUAUGCC 130 2075-2096 AD-69988 A-140469GUGAAAAUCACCUAUGAAGAA  45 2137-2156 A-140470 UUCUUCAUAGGUGAUUUUCACCC 1312135-2156 AD-70034 A-140561 GUGAAAAUCACCUAUGAAGAA  46 2137-2156 A-140562UUCUUCAUAGGUGAUUUUCACCC 132 2135-2156 AD-69996 A-140485ACAAUUGAGGAUGCUAUAAAA  47 2173- A-140486 UUUUAUAGCAUCCUCAAUUGUGA 1332171- 2192_G21A 2192_G21A AD-70042 A-140577 ACAAUUGAGGAUGCUAUAAAA  482173- A-140578 UUUUAUAGCAUCCUCAAUUGUGA 134 2171- 2192_G21A 2192_G21AAD-69977 A-140447 UGCUAUAAAGAACAACUCCUU  49 2184-2203 A-140448AAGGAGUUGUUCUUUAUAGCAUC 135 2182-2203 AD-70023 A-140539UGCUAUAAAGAACAACUCCUU  50 2184-2203 A-140540 AAGGAGUUGUUCUUUAUAGCAUC 1362182-2203 AD-69978 A-140449 GCUAUAAAGAACAACUCCUUU  51 2185-2204 A-140450AAAGGAGUUGUUCUUUAUAGCAU 137 2183-2204 AD-70024 A-140541GCUAUAAAGAACAACUCCUUU  52 2185-2204 A-140542 AAAGGAGUUGUUCUUUAUAGCAU 1382183-2204 AD-69972 A-140437 UAUAAAGAACAACUCCUUUUA  53 2187-2206 A-140438UAAAAGGAGUUGUUCUUUAUAGC 139 2185-2206 AD-70018 A-140529UAUAAAGAACAACUCCUUUUA  54 2187-2206 A-140530 UAAAAGGAGUUGUUCUUUAUAGC 1402185-2206 AD-69971 A-140435 UAAAGAACAACUCCUUUUAUA  55 2189- A-140436UAUAAAAGGAGUUGUUCUUUAUA 141 2187- 2208_G21A 2208_G21A AD-70017 A-140527UAAAGAACAACUCCUUUUAUA  56 2189- A-140528 UAUAAAAGGAGUUGUUCUUUAUA 1422187- 2208_G21A 2208_G21A AD-69994 A-140481 GACAUGCUGAUAACUGGUGGA  572578- A-140482 UCCACCAGUUAUCAGCAUGUCCU 143 2576- 2597_C21A 2597_C21AAD-70040 A-140573 GACAUGCUGAUAACUGGUGGA  58 2578- A-140574UCCACCAGUUAUCAGCAUGUCCU 144 2576- 2597_C21A 2597_C21A AD-69985 A-140463AUGCUGAUAACUGGUGGCAGA  59 2581-2600 A-140464 UCUGCCACCAGUUAUCAGCAUGU 1452579-2600 AD-70031 A-140555 AUGCUGAUAACUGGUGGCAGA  60 2581-2600 A-140556UCUGCCACCAGUUAUCAGCAUGU 146 2579-2600 AD-69982 A-140457UGAUAACUGGUGGCAGACAUA  61 2585- A-140458 UAUGUCUGCCACCAGUUAUCAGC 1472583- 2604_C21A 2604_C21A AD-70028 A-140549 UGAUAACUGGUGGCAGACAUA  622585- A-140550 UAUGUCUGCCACCAGUUAUCAGC 148 2583- 2604_C21A 2604_C21AAD-69984 A-140461 UACAAGGUUGGCUUCAUGAAA  63 2620- A-140462UUUCAUGAAGCCAACCUUGUAUC 149 2618- 2639_G21A 2639_G21A AD-70030 A-140553UACAAGGUUGGCUUCAUGAAA  64 2620- A-140554 UUUCAUGAAGCCAACCUUGUAUC 1502618- 2639_G21A 2639_G21A AD-69991 A-140475 ACAAGGUUGGCUUCAUGAAGA  652621-2640 A-140476 UCUUCAUGAAGCCAACCUUGUAU 151 2619-2640 AD-70037A-140567 ACAAGGUUGGCUUCAUGAAGA  66 2621-2640 A-140568UCUUCAUGAAGCCAACCUUGUAU 152 2619-2640 AD-70005 A-140503AAGCUUGAGGGUUUCACCUUA  67 2956- A-140504 UAAGGUGAAACCCUCAAGCUUCU 1532954- 2975_G21A 2975_G21A AD-70051 A-140595 AAGCUUGAGGGUUUCACCUUA  682956- A-140596 UAAGGUGAAACCCUCAAGCUUCU 154 2954- 2975_G21A 2975_G21AAD-70002 A-140497 AAGAGUGAGGUUGACAAGUUA  69 3025- A-140498UAACUUGUCAACCUCACUCUUCC 155 3023- 3044_C21A 3044_C21A AD-70048 A-140589AAGAGUGAGGUUGACAAGUUA  70 3025- A-140590 UAACUUGUCAACCUCACUCUUCC 1563023- 3044_C21A 3044_C21A AD-69992 A-140477 GUUCAACAAGGAGAAUUGUUA  713042- A-140478 UAACAAUUCUCCUUGUUGAACUU 157 3040- 3061_G21A 3061_G21AAD-70038 A-140569 GUUCAACAAGGAGAAUUGUUA  72 3042- A-140570UAACAAUUCUCCUUGUUGAACUU 158 3040- 3061_G21A 3061_G21A AD-69993 A-140479ACAAGGAGAAUUGUUGGAAAA  73 3047-3066 A-140480 UUUUCCAACAAUUCUCCUUGUUG 1593045-3066 AD-70039 A-140571 ACAAGGAGAAUUGUUGGAAAA  74 3047-3066 A-140572UUUUCCAACAAUUCUCCUUGUUG 160 3045-3066 AD-69980 A-140453ACCAAGUUUGGAAUAAGCUUU  75 3091-3110 A-140454 AAAGCUUAUUCCAAACUUGGUGG 1613089-3110 AD-70026 A-140545 ACCAAGUUUGGAAUAAGCUUU  76 3091-3110 A-140546AAAGCUUAUUCCAAACUUGGUGG 162 3089-3110 AD-69979 A-140451UAUCUUCUUUGCCAUCAAAGA  77 3891-3910 A-140452 UCUUUGAUGGCAAAGAAGAUAGA 1633889-3910 AD-70025 A-140543 UAUCUUCUUUGCCAUCAAAGA  78 3891-3910 A-140544UCUUUGAUGGCAAAGAAGAUAGA 164 3889-3910 AD-69974 A-140441UCUUCUUUGCCAUCAAAGAUA  79 3893- A-140442 UAUCUUUGAUGGCAAAGAAGAUA 1653891- 3912_G21A 3912_G21A AD-70020 A-140533 UCUUCUUUGCCAUCAAAGAUA  803893- A-140534 UAUCUUUGAUGGCAAAGAAGAUA 166 3891- 3912_G21A 3912_G21AAD-70004 A-140501 CAGAACAUGGAUCUAUUAAAA  81 4152- A-140502UUUUAAUAGAUCCAUGUUCUGUG 167 4150- 4171_G21A 4171_G21A AD-70050 A-140593CAGAACAUGGAUCUAUUAAAA  82 4152- A-140594 UUUUAAUAGAUCCAUGUUCUGUG 1684150- 4171_G21A 4171_G21A AD-70009 A-140511 ACAAUGAUAAGCAAAUUCAAA  834266-4285 A-140512 UUUGAAUUUGCUUAUCAUUGUGU 169 4264-4285 AD-70055A-140603 ACAAUGAUAAGCAAAUUCAAA  84 4266-4285 A-140604UUUGAAUUUGCUUAUCAUUGUGU 170 4264-4285 AD-69998 A-140489AAUGGUGAAUAUGCAAUUAGA  85 4300- A-140490 UCUAAUUGCAUAUUCACCAUUUA 1714298- 4319_G21A 4319_G21A AD-70044 A-140581 AAUGGUGAAUAUGCAAUUAGA  864300- A-140582 UCUAAUUGCAUAUUCACCAUUUA 172 4298- 4319_G21A 4319_G21AAD-70006 A-140505 ACCAAUGAACAGCAAAGCAUA  87 4519-4538 A-140506UAUGCUUUGCUGUUCAUUGGUUU 173 4517-4538 AD-70052 A-140597ACCAAUGAACAGCAAAGCAUA  88 4519-4538 A-140598 UAUGCUUUGCUGUUCAUUGGUUU 1744517-4538 AD-70001 A-140495 CCAUCUUUGAAUCAUUGGAAA  89 4599-4618 A-140496UUUCCAAUGAUUCAAAGAUGGUU 175 4597-4618 AD-70047 A-140587CCAUCUUUGAAUCAUUGGAAA  90 4599-4618 A-140588 UUUCCAAUGAUUCAAAGAUGGUU 1764597-4618 AD-69997 A-140487 AAGAAUAAAGAAUGAAACAAA  91 4618-4637 A-140488UUUGUUUCAUUCUUUAUUCUUUC 177 4616-4637 AD-70043 A-140579AAGAAUAAAGAAUGAAACAAA  92 4618-4637 A-140580 UUUGUUUCAUUCUUUAUUCUUUC 1784616-4637 AD-69999 A-140491 AAUAAAGAAUGAAACAAAUUA  93 4621- A-140492UAAUUUGUUUCAUUCUUUAUUCU 179 4619- 4640_C21A 4640_C21A AD-70045 A-140583AAUAAAGAAUGAAACAAAUUA  94 4621- A-140584 UAAUUUGUUUCAUUCUUUAUUCU 1804619- 4640_C21A 4640_C21A AD-70008 A-140509 AUCCAACCAACUCAAUUAUUA  954703- A-140510 UAAUAAUUGAGUUGGUUGGAUUU 181 4701- 4722_G21A 4722_G21AAD-70054 A-140601 AUCCAACCAACUCAAUUAUUA  96 4703- A-140602UAAUAAUUGAGUUGGUUGGAUUU 182 4701- 4722_G21A 4722_G21A AD-70000 A-140493CACUGUAUAAAUCCAACCUUA  97 5599- A-140494 UAAGGUUGGAUUUAUACAGUGAA 1835597- 5618_C21A 5618_C21A AD-70046 A-140585 CACUGUAUAAAUCCAACCUUA  985599- A-140586 UAAGGUUGGAUUUAUACAGUGAA 184 5597- 5618_C21A 5618_C21AAD-70015 A-140523 AACUGUGGAAGGCAUAGGAAA  99  337-356 A-140524UUUCCUAUGCCUUCCACAGUUGU 185  335-356 AD-70012 A-140517GGAUUUCAAACCUUUAGAUCA 100  673- A-140518 UGAUCUAAAGGUUUGAAAUCCUC 186 671- 692_C21A 692_C21A AD-70014 A-140521 ACAGAGAUAGGCAUUGAAAUA 101 860- A-140522 UAUUUCAAUGCCUAUCUCUGUGU 187  858- 879_G21A 879_G21AAD-70010 A-140513 GGCAUUGAAAUGAAAUUUAAA 102  869-888 A-140514UUUAAAUUUCAUUUCAAUGCCUA 188  867-888 AD-70013 A-140519ACAAUCCAGGAUGCUAUAAAA 103 2168- A-140520 UUUUAUAGCAUCCUGGAUUGUGA 1892166- 2187_G21A 2187_G21A AD-70011 A-140515 CAAGAUGGAAGUGGAGAAAUU 1043019-3038 A-140516 AAUUUCUCCACUUCCAUCUUGCG 190 3017-3038

TABLE 4 XDH Modified Sequences Anti- Sense SEQ Start in sense SEQStart in Duplex Oligo Sense Sequence ID NM_ Oligo Antisense Sequence IDNM_ Name Name (5′ to 3′) NO 000379.3 Name (5′ to 3′) NO 000379.3 AD-A-140499 AACUGUGGAAGGAAUAGGAAAdTdT 191  339 A-140500UUUCCUAUUCCUUCCACAGUUGUdTdT 277  337 70003 AD- A-140591asascuguGfgAfAfGfgaauagga 192  339 A-140592 usUfsuccUfaUfUfccuuCfcAfcag278  337 70049 aaL96 uusgsu AD- A-140455 GAGGAGAUUGAGAAUGCCUUAdTdT 193 490 A-140456 UAAGGCAUUCUCAAUCUCCUCCAdTdT 279  488 69981 AD- A-140547gsasggagAfuUfGfAfgaaugccu 194  490 A-140548 usAfsaggCfaUfUfcucaAfuCfucc280  488 70027 uaL96 ucscsa AD- A-140433 GAGAUUGAGAAUGCCUUCCAAdTdT 195 493 A-140434 UUGGAAGGCAUUCUCAAUCUCCUdTdT 281  491 69970 AD- A-140525gsasgauuGfaGfAfAfugccuucc 196  493 A-140526 usUfsggaAfgGfCfauucUfcAfauc282  491 70016 aaL96 ucscsu AD- A-140507 GUUCAAGAAUAUGCUGUUUCAdTdT 197 888 A-140508 UGAAACAGCAUAUUCUUGAACUUdTdT 283  886 70007 AD- A-140599gsusucaaGfaAfUfAfugcuguuu 198  888 A-140600 usGfsaaaCfaGfCfauauUfcUfuga284  886 70053 caL96 acsusu AD- A-140483 AAAAGACAGAGGUGUUCAGAAdTdT 1991046 A-140484 UUCUGAACACCUCUGUCUUUUGGdTdT 285 1044 69995 AD- A-140575asasaagaCfaGfAfGfguguucag 200 1046 A-140576 usUfscugAfaCfAfccucUfgUfcuu286 1044 70041 aaL96 uusgsg AD- A-140439 AAAGACAGAGGUGUUCAGAGAdTdT 2011047 A-140440 UCUCUGAACACCUCUGUCUUUUGdTdT 287 1045 69973 AD- A-140531asasagacAfgAfGfGfuguucaga 202 1047 A-140532 usCfsucuGfaAfCfaccuCfuGfucu288 1045 70019 gaL96 uususg AD- A-140443 CGGAGAGAAGAUGACAUUGCAdTdT 2031357 A-140444 UGCAAUGUCAUCUUCUCUCCGGGdTdT 289 1355 69975 AD- A-140535csgsgagaGfaAfGfAfugacauug 204 1357 A-140536 usGfscaaUfgUfCfaucuUfcUfcuc290 1355 70021 caL96 cgsgsg AD- A-140459 AGAGAAGAUGACAUUGCCAAAdTdT 2051360 A-140460 UUUGGCAAUGUCAUCUUCUCUCCdTdT 291 1358 69983 AD- A-140551asgsagaaGfaUfGfAfcauugcca 206 1360 A-140552 usUfsuggCfaAfUfgucaUfcUfucu292 1358 70029 aaL96 cuscsc AD- A-140465 GAAGAUGACAUUGCCAAGGUAdTdT 2071363 A-140466 UACCUUGGCAAUGUCAUCUUCUCdTdT 293 1361 69986 AD- A-140557gsasagauGfaCfAfUfugccaagg 208 1363 A-140558 usAfsccuUfgGfCfaaugUfcAfucu294 1361 70032 uaL96 ucsusc AD- A-140445 CAGUCUGAGGAGGACAUGGUAdTdT 2091783 A-140446 UACCAUGUCCUCCUCAGACUGACdTdT 295 1781 69976 AD- A-140537csasgucuGfaGfGfAfggacaugg 210 1783 A-140538 usAfsccaUfgUfCfcuccUfcAfgac296 1781 70022 uaL96 ugsasc AD- A-140467 AAGGAUAAGGUUACUUGUGUUdTdT 2112053 A-140468 AACACAAGUAACCUUAUCCUUCGdTdT 297 2051 69987 AD- A-140559asasggauAfaGfGfUfuacuugug 212 2053 A-140560 asAfscacAfaGfUfaaccUfuAfucc298 2051 70033 uuL96 uuscsg AD- A-140473 AGGAUAAGGUUACUUGUGUUAdTdT 2132054 A-140474 UAACACAAGUAACCUUAUCCUUCdTdT 299 2052 69990 AD- A-140565asgsgauaAfgGfUfUfacuugugu 214 2054 A-140566 usAfsacaCfaAfGfuaacCfuUfauc300 2052 70036 uaL96 cususc AD- A-140471 CAUAUCAUUGGUGCUGUGGUUdTdT 2152077 A-140472 AACCACAGCACCAAUGAUAUGCCdTdT 301 2075 69989 AD- A-140563csasuaucAfuUfGfGfugcugugg 216 2077 A-140564 asAfsccaCfaGfCfaccaAfuGfaua302 2075 70035 uuL96 ugscsc AD- A-140469 GUGAAAAUCACCUAUGAAGAAdTdT 2172137 A-140470 UUCUUCAUAGGUGAUUUUCACCCdTdT 303 2135 69988 AD- A-140561gsusgaaaAfuCfAfCfcuaugaag 218 2137 A-140562 usUfscuuCfaUfAfggugAfuUfuuc304 2135 70034 aaL96 acscsc AD- A-140485 ACAAUUGAGGAUGCUAUAAAAdTdT 2192173 A-140486 UUUUAUAGCAUCCUCAAUUGUGAdTdT 305 2171 69996 AD- A-140577ascsaauuGfaGfGfAfugcuauaa 220 2173 A-140578 usUfsuuaUfaGfCfauccUfcAfauu306 2171 70042 aaL96 gusgsa AD- A-140447 UGCUAUAAAGAACAACUCCUUdTdT 2212184 A-140448 AAGGAGUUGUUCUUUAUAGCAUCdTdT 307 2182 69977 AD- A-140539usgscuauAfaAfGfAfacaacucc 222 2184 A-140540 asAfsggaGfuUfGfuucuUfuAfuag308 2182 70023 uuL96 casusc AD- A-140449 GCUAUAAAGAACAACUCCUUUdTdT 2232185 A-140450 AAAGGAGUUGUUCUUUAUAGCAUdTdT 309 2183 69978 AD- A-140541gscsuauaAfaGfAfAfcaacuccu 224 2185 A-140542 asAfsaggAfgUfUfguucUfuUfaua310 2183 70024 uuL96 gcsasu AD- A-140437 UAUAAAGAACAACUCCUUUUAdTdT 2252187 A-140438 UAAAAGGAGUUGUUCUUUAUAGCdTdT 311 2185 69972 AD- A-140529usasuaaaGfaAfCfAfacuccuuu 226 2187 A-140530 usAfsaaaGfgAfGfuuguUfcUfuua312 2185 70018 uaL96 uasgsc AD- A-140435 UAAAGAACAACUCCUUUUAUAdTdT 2272189 A-140436 UAUAAAAGGAGUUGUUCUUUAUAdTdT 313 2187 69971 AD- A-140527usasaagaAfcAfAfCfuccuuuua 228 2189 A-140528 usAfsuaaAfaGfGfaguuGfuUfcuu314 2187 70017 uaL96 uasusa AD- A-140481 GACAUGCUGAUAACUGGUGGAdTdT 2292578 A-140482 UCCACCAGUUAUCAGCAUGUCCUdTdT 315 2576 69994 AD- A-140573gsascaugCfuGfAfUfaacuggug 230 2578 A-140574 usCfscacCfaGfUfuaucAfgCfaug316 2576 70040 gaL96 ucscsu AD- A-140463 AUGCUGAUAACUGGUGGCAGAdTdT 2312581 A-140464 UCUGCCACCAGUUAUCAGCAUGUdTdT 317 2579 69985 AD- A-140555asusgcugAfuAfAfCfugguggca 232 2581 A-140556 usCfsugcCfaCfCfaguuAfuCfagc318 2579 70031 gaL96 ausgsu AD- A-140457 UGAUAACUGGUGGCAGACAUAdTdT 2332585 A-140458 UAUGUCUGCCACCAGUUAUCAGCdTdT 319 2583 69982 AD- A-140549usgsauaaCfuGfGfUfggcagaca 234 2585 A-140550 usAfsuguCfuGfCfcaccAfgUfuau320 2583 70028 uaL96 casgsc AD- A-140461 UACAAGGUUGGCUUCAUGAAAdTdT 2352620 A-140462 UUUCAUGAAGCCAACCUUGUAUCdTdT 321 2618 69984 AD- A-140553usascaagGfuUfGfGfcuucauga 236 2620 A-140554 usUfsucaUfgAfAfgccaAfcCfuug322 2618 70030 aaL96 uasusc AD- A-140475 ACAAGGUUGGCUUCAUGAAGAdTdT 2372621 A-140476 UCUUCAUGAAGCCAACCUUGUAUdTdT 323 2619 69991 AD- A-140567ascsaaggUfuGfGfCfuucaugaa 238 2621 A-140568 usCfsuucAfuGfAfagccAfaCfcuu324 2619 70037 gaL96 gusasu AD- A-140503 AAGCUUGAGGGUUUCACCUUAdTdT 2392956 A-140504 UAAGGUGAAACCCUCAAGCUUCUdTdT 325 2954 70005 AD- A-140595asasgcuuGfaGfGfGfuuucaccu 240 2956 A-140596 usAfsaggUfgAfAfacccUfcAfagc326 2954 70051 uaL96 uuscsu AD- A-140497 AAGAGUGAGGUUGACAAGUUAdTdT 2413025 A-140498 UAACUUGUCAACCUCACUCUUCCdTdT 327 3023 70002 AD- A-140589asasgaguGfaGfGfUfugacaagu 242 3025 A-140590 usAfsacuUfgUfCfaaccUfcAfcuc328 3023 70048 uaL96 uuscsc AD- A-140477 GUUCAACAAGGAGAAUUGUUAdTdT 2433042 A-140478 UAACAAUUCUCCUUGUUGAACUUdTdT 329 3040 69992 AD- A-140569gsusucaaCfaAfGfGfagaauugu 244 3042 A-140570 usAfsacaAfuUfCfuccuUfgUfuga330 3040 70038 uaL96 acsusu AD- A-140479 ACAAGGAGAAUUGUUGGAAAAdTdT 2453047 A-140480 UUUUCCAACAAUUCUCCUUGUUGdTdT 331 3045 69993 AD- A-140571ascsaaggAfgAfAfUfuguuggaa 246 3047 A-140572 usUfsuucCfaAfCfaauuCfuCfcuu332 3045 70039 aaL96 gususg AD- A-140453 ACCAAGUUUGGAAUAAGCUUUdTdT 2473091 A-140454 AAAGCUUAUUCCAAACUUGGUGGdTdT 333 3089 69980 AD- A-140545ascscaagUfuUfGfGfaauaagcu 248 3091 A-140546 asAfsagcUfuAfUfuccaAfaCfuug334 3089 70026 uuL96 gusgsg AD- A-140451 UAUCUUCUUUGCCAUCAAAGAdTdT 2493891 A-140452 UCUUUGAUGGCAAAGAAGAUAGAdTdT 335 3889 69979 AD- A-140543usasucuuCfuUfUfGfccaucaaa 250 3891 A-140544 usCfsuuuGfaUfGfgcaaAfgAfaga336 3889 70025 gaL96 uasgsa AD- A-140441 UCUUCUUUGCCAUCAAAGAUAdTdT 2513893 A-140442 UAUCUUUGAUGGCAAAGAAGAUAdTdT 337 3891 69974 AD- A-140533uscsuucuUfuGfCfCfaucaaaga 252 3893 A-140534 usAfsucuUfuGfAfuggcAfaAfgaa338 3891 70020 uaL96 gasusa AD- A-140501 CAGAACAUGGAUCUAUUAAAAdTdT 2534152 A-140502 UUUUAAUAGAUCCAUGUUCUGUGdTdT 339 4150 70004 AD- A-140593csasgaacAfuGfGfAfucuauuaa 254 4152 A-140594 usUfsuuaAfuAfGfauccAfuGfuuc340 4150 70050 aaL96 ugsusg AD- A-140511 ACAAUGAUAAGCAAAUUCAAAdTdT 2554266 A-140512 UUUGAAUUUGCUUAUCAUUGUGUdTdT 341 4264 70009 AD- A-140603ascsaaugAfuAfAfGfcaaauuca 256 4266 A-140604 usUfsugaAfuUfUfgcuuAfuCfauu342 4264 70055 aaL96 gusgsu AD- A-140489 AAUGGUGAAUAUGCAAUUAGAdTdT 2574300 A-140490 UCUAAUUGCAUAUUCACCAUUUAdTdT 343 4298 69998 AD- A-140581asasugguGfaAfUfAfugcaauua 258 4300 A-140582 usCfsuaaUfuGfCfauauUfcAfcca344 4298 70044 gaL96 uususa AD- A-140505 ACCAAUGAACAGCAAAGCAUAdTdT 2594519 A-140506 UAUGCUUUGCUGUUCAUUGGUUUdTdT 345 4517 70006 AD- A-140597ascscaauGfaAfCfAfgcaaagca 260 4519 A-140598 usAfsugcUfuUfGfcuguUfcAfuug346 4517 70052 uaL96 gususu AD- A-140495 CCAUCUUUGAAUCAUUGGAAAdTdT 2614599 A-140496 UUUCCAAUGAUUCAAAGAUGGUUdTdT 347 4597 70001 AD- A-140587cscsaucuUfuGfAfAfucauugga 262 4599 A-140588 usUfsuccAfaUfGfauucAfaAfgau348 4597 70047 aaL96 ggsusu AD- A-140487 AAGAAUAAAGAAUGAAACAAAdTdT 2634618 A-140488 UUUGUUUCAUUCUUUAUUCUUUCdTdT 349 4616 69997 AD- A-140579asasgaauAfaAfGfAfaugaaaca 264 4618 A-140580 usUfsuguUfuCfAfuucuUfuAfuuc350 4616 70043 aaL96 uususc AD- A-140491 AAUAAAGAAUGAAACAAAUUAdTdT 2654621 A-140492 UAAUUUGUUUCAUUCUUUAUUCUdTdT 351 4619 69999 AD- A-140583asasuaaaGfaAfUfGfaaacaaau 266 4621 A-140584 usAfsauuUfgUfUfucauUfcUfuua352 4619 70045 uaL96 uuscsu AD- A-140509 AUCCAACCAACUCAAUUAUUAdTdT 2674703 A-140510 UAAUAAUUGAGUUGGUUGGAUUUdTdT 353 4701 70008 AD- A-140601asusccaaCfcAfAfCfucaauuau 268 4703 A-140602 usAfsauaAfuUfGfaguuGfgUfugg354 4701 70054 uaL96 aususu AD- A-140493 CACUGUAUAAAUCCAACCUUAdTdT 2695599 A-140494 UAAGGUUGGAUUUAUACAGUGAAdTdT 355 5597 70000 AD- A-140585csascuguAfuAfAfAfuccaaccu 270 5599 A-140586 usAfsaggUfuGfGfauuuAfuAfcag356 5597 70046 uaL96 ugsasa AD- A-140523 AACUGUGGAAGGCAUAGGAAAdTdT 271 337 A-140524 UUUCCUAUGCCUUCCACAGUUGUdTdT 357  335 70015 AD- A-140517GGAUUUCAAACCUUUAGAUCAdTdT 272  673 A-140518 UGAUCUAAAGGUUUGAAAUCCUCdTdT358  671 70012 AD- A-140521 ACAGAGAUAGGCAUUGAAAUAdTdT 273  860 A-140522UAUUUCAAUGCCUAUCUCUGUGUdTdT 359  858 70014 AD- A-140513GGCAUUGAAAUGAAAUUUAAAdTdT 274  869 A-140514 UUUAAAUUUCAUUUCAAUGCCUAdTdT360  867 70010 AD- A-140519 ACAAUCCAGGAUGCUAUAAAAdTdT 275 2168 A-140520UUUUAUAGCAUCCUGGAUUGUGAdTdT 361 2166 70013 AD- A-140515CAAGAUGGAAGUGGAGAAAUUdTdT 276 3019 A-140516 AAUUUCUCCACUUCCAUCUUGCGdTdT362 3017 70011

TABLE 5 Single dose screen in Primary Cynomolgus Hepatocytes and PrimaryMouse Hepatocytes Primary Cynomolgus Hepatocytes Primary MouseHepatocytes 10 nM 10 nM 0.1 nM 0.1 nM 10 nM 10 nM 0.1 nM 0.1 nM DuplexID Avg SD Avg SD Avg SD Avg SD AD-70016 34.3 6.9 41.7 5.2 4.5 0.6 38.13.4 AD-70017 65.5 5.0 66.2 4.2 8.0 2.5 73.6 7.1 AD-70018 36.3 0.9 74.918.3 5.9 1.1 49.6 6.7 AD-70019 59.4 9.2 62.9 13.3 19.8 0.5 123.9 18.8AD-70020 40.5 7.0 38.9 6.6 10.7 0.8 122.7 24.0 AD-70021 41.0 2.1 72.57.6 13.7 1.5 88.1 5.8 AD-70022 44.8 5.9 49.7 19.0 17.3 2.3 105.7 7.6AD-70023 37.2 9.0 71.7 11.0 5.6 1.7 30.6 5.9 AD-70024 61.0 10.5 86.4 9.37.2 0.6 32.6 4.0 AD-70025 40.9 9.2 80.1 9.1 35.3 3.8 109.3 4.9 AD-7002641.1 5.7 54.1 8.1 7.3 1.1 57.8 4.8 AD-70027 39.0 5.5 57.9 7.2 11.8 1.6102.4 18.8 AD-70028 47.1 2.6 55.1 15.9 11.0 2.2 95.9 17.2 AD-70029 41.57.5 62.7 9.8 8.3 2.0 63.5 11.2 AD-70030 42.4 5.9 45.2 11.0 16.6 1.8101.1 7.5 AD-70031 52.4 2.2 67.8 20.1 38.0 4.6 99.5 11.6 AD-70032 51.16.0 74.3 14.9 22.4 9.9 100.9 12.8 AD-70033 19.6 6.7 45.7 7.5 6.2 0.827.2 6.9 AD-70034 39.0 9.1 88.9 22.1 16.2 3.8 89.2 6.2 AD-70035 37.416.3 84.0 6.5 16.1 1.7 96.5 7.7 AD-70036 53.0 5.9 61.3 11.3 49.8 7.1105.9 8.4 AD-70037 46.3 9.1 102.4 23.5 17.1 3.5 97.7 12.5 AD-70038 49.03.6 54.3 17.6 86.4 5.3 95.6 11.5 AD-70039 56.3 10.5 85.4 11.4 75.0 5.8101.6 10.3 AD-70040 52.8 3.5 101.1 17.9 95.9 5.1 96.2 11.1 AD-70041 66.520.5 85.7 12.1 48.7 3.0 97.7 5.3 AD-70042 34.8 4.6 47.4 4.2 20.3 1.1106.8 11.1 AD-70043 42.0 4.7 85.8 22.5 117.6 19.4 113.4 24.1 AD-7004437.5 9.6 47.1 22.7 87.4 13.6 111.6 16.7 AD-70045 50.9 4.8 84.4 18.3 95.55.4 108.8 5.4 AD-70046 81.3 20.3 61.2 21.6 95.5 5.4 111.8 18.5 AD-7004759.7 12.4 82.7 28.2 100.0 9.7 93.2 14.2 AD-70048 46.7 8.6 61.3 10.3 96.811.9 109.7 23.7 AD-70049 33.8 10.8 76.7 15.3 20.4 5.7 95.4 6.8 AD-7005027.1 4.7 57.7 4.9 107.8 11.0 101.1 5.2 AD-70051 27.6 7.3 76.8 6.7 101.514.5 99.1 12.7 AD-70052 36.2 13.1 37.6 14.8 110.3 11.4 109.3 14.8AD-70053 26.7 5.9 86.9 15.1 104.6 21.4 100.9 9.7 AD-70055 36.5 7.6 58.916.7 105.0 14.5 98.7 8.0 AD-69970 47.5 4.5 48.5 7.1 6.1 0.9 22.5 7.7AD-69971 60.2 11.6 88.4 25.0 10.1 4.8 33.2 10.4 AD-69972 52.8 4.5 67.817.0 6.5 1.9 25.5 10.9 AD-69973 36.2 5.3 64.5 13.0 46.6 13.2 50.1 13.9AD-69974 30.3 3.5 60.8 11.2 9.1 1.5 44.8 11.0 AD-69975 29.7 3.2 48.6 8.212.5 6.2 20.3 3.2 AD-69976 40.4 6.0 67.6 5.4 10.4 2.3 45.9 10.3 AD-6997745.7 7.1 91.6 12.5 6.3 0.9 19.7 3.4 AD-69979 25.8 4.3 58.0 7.1 14.6 9.837.8 2.9 AD-69980 30.9 7.9 51.6 13.5 9.5 2.6 23.1 6.3 AD-69981 37.0 6.044.2 9.2 14.2 10.8 47.8 3.0 AD-69982 22.6 2.8 48.4 10.7 9.3 1.2 30.410.0 AD-69983 31.1 3.6 49.7 10.7 7.9 0.8 27.8 5.6 AD-69984 39.7 6.8 66.213.0 49.0 1.1 87.8 13.2 AD-69985 39.2 4.5 47.7 5.4 14.0 3.9 40.0 8.3AD-69986 21.0 8.3 55.4 7.9 6.8 0.7 24.0 0.7 AD-69987 18.6 6.0 49.4 10.011.5 6.0 14.3 3.6 AD-69988 29.5 3.1 56.4 9.9 10.7 3.7 17.2 5.5 AD-6998928.4 7.8 61.5 8.3 13.3 7.5 22.9 5.7 AD-69990 28.4 4.0 66.5 18.3 13.5 7.946.4 8.6 AD-69991 37.4 8.1 48.6 3.8 12.4 1.3 52.4 4.5 AD-69992 31.3 5.663.3 23.3 117.7 27.8 101.8 4.3 AD-69993 40.4 3.1 54.4 5.3 86.3 12.9 86.79.5 AD-69994 30.1 7.5 60.5 5.5 46.0 15.2 78.7 11.6 AD-69995 51.9 7.698.7 22.7 17.0 2.3 72.0 3.2 AD-69996 34.3 4.5 43.2 4.9 72.1 14.5 89.49.5 AD-69998 37.3 4.3 64.0 2.8 120.6 15.1 105.5 15.8 AD-69999 60.4 13.097.6 20.1 122.9 40.3 92.6 12.9 AD-70000 74.5 11.1 84.3 9.5 120.4 15.497.9 27.4 AD-70001 39.8 5.9 73.5 11.2 121.6 16.8 101.7 19.2 AD-7000235.7 3.3 63.0 6.9 102.2 8.7 84.2 15.9 AD-70003 26.4 4.3 55.7 5.1 9.8 3.343.1 11.1 AD-70004 42.1 5.9 58.1 15.0 130.1 45.5 104.0 9.4 AD-70005 25.72.5 53.8 5.4 146.4 47.4 102.7 18.9 AD-70006 31.6 3.7 50.4 1.5 110.2 19.2106.4 15.4 AD-70007 31.1 4.4 49.5 4.9 120.1 16.3 89.2 3.0 AD-70008 40.23.6 64.4 3.2 106.1 13.1 105.6 22.0 AD-70009 39.7 5.4 63.7 3.5 112.1 8.198.7 20.3 AD-70010 96.3 26.2 110.6 11.7 5.2 1.4 10.2 3.4 AD-70011 77.010.7 102.9 12.6 10.3 1.1 15.9 2.0 AD-70012 92.4 23.9 95.6 7.4 8.1 1.416.1 2.7 AD-70013 44.8 9.2 79.9 2.3 6.7 0.8 14.3 3.0 AD-70014 84.6 17.788.4 9.5 6.9 1.6 16.2 2.8 AD-70015 48.4 14.9 80.8 6.2 8.0 2.2 14.5 2.4

Example 3. iRNA Synthesis and in Vitro Screening in Primary HumanHepatocytes

siRNA Design

A set of siRNAs targeting human XDH “xanthine dehydrogenase” gene (humanNCBI refseqID: NM_000379; NCBI GeneID: 7498) were designed using customR and Python scripts. The human XDH REFSEQ mRNA has a length of 5717bases. The rationale and method for the set of siRNA designs is asfollows: the predicted efficacy for every potential 19mer siRNA fromposition 10 through position 5717 was determined with a linear modelderived the direct measure of mRNA knockdown from more than 20,000distinct siRNA designs targeting a large number of vertebrate genes. Thecustom Python script built the set of siRNAs by systematically selectinga siRNA every 11 bases along the target mRNA starting at position 10. Ateach of the positions, the neighboring siRNA (one position to the 5′ endof the mRNA, one position to the 3′ end of the mRNA) was swapped intothe design set if the predicted efficacy was better than the efficacy atthe exact every-11th siRNA. Low complexity siRNAs, i.e., those withShannon Entropy measures below 1.35 were excluded from the set.

A detailed list of the unmodified XDH sense and antisense strandsequences is shown in Table 6 and a detailed list of the modified XDHsense and antisense strand sequences is shown in Table 7.

Cell Culture and Transfections

Primary human hepatocytes were thawed and cultured in WMEM with 5% FBSand maintenance reagents (Invitrogen, Carlsbad Calif. Cat #CM3000) oncollagen-coated plates. After 24 hours, media was removed and replacedwith 40 μl of WMEM with maintenance reagents (Inivtrogen, CarlsbadCalif. Cat #CM4000) containing ˜5×10³ cells. Separately, 7.35 μl ofOpti-MEM plus 0.15 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) was added to 7.5 μl of siRNA duplexe andincubated at room temperature for 20 minutes. Ten μl of lipoplex mixturewas then transferred to the cells. Cells were incubated for 24 hoursprior to RNA purification. Single dose experiments were performed at 10nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 50 μl ofLysis/Binding Buffer and 25 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μL Elution Buffer, re-capturedand supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 hours at 37° C.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μL of GAPDHTaqMan Probe (4326317E), 0.5 μl XDH probe (Hs00166010_ml) and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384 well plates (Roche cat #04887301001). Real time PCR was done in aLightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay.Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data was analyzed using theΔΔCt method and normalized to assays performed with mock transfectedcells.

Table 8 shows the results of a single dose screen in Primary HumanHepatocytes transfected with the indicated iRNAs.

TABLE 6 XDH Unmodified Sequences Anti- Sense SEQ sense SEQ Duplex OligoSense Sequence ID Position in Oligo Antisense Sequence ID Position inName Name (5′ to 3′) NO NM_000379.3 Name (5′-3′) NO NM_000379.3 AD-71930A-143855 ACUACCUGCCAGUGUCUCU 363 18-36 A-143856 AGAGACACUGGCAGGUAGU 67718-36 AD-71931 A-143857 UUAGGAGUGAGGUACCUGA 364 36-54 A-143858UCAGGUACCUCACUCCUAA 678 36-54 AD-71932 A-143861 AACCUGUGACAAUGACAGA 36569-87 A-143862 UCUGUCAUUGUCACAGGUU 679 69-87 AD-71933 A-143863GCAGACAAAUUGGUUUUCU 366  86-104 A-143864 AGAAAACCAAUUUGUCUGC 680  86-104AD-71934 A-143865 UUUGUGAAUGGCAGAAAGA 367 104-122 A-143866UCUUUCUGCCAUUCACAAA 681 104-122 AD-71935 A-143867 AAGGUGGUGGAGAAAAAUA368 119-137 A-143868 UAUUUUUCUCCACCACCUU 682 119-137 AD-71936 A-143871CCUUUUGGCCUACCUGAGA 369 154-172 A-143872 UCUCAGGUAGGCCAAAAGG 683 154-172AD-71937 A-143873 AAGAAAGUUGGGGCUGAGU 370 172-190 A-143874ACUCAGCCCCAACUUUCUU 684 172-190 AD-71938 A-143875 AGUGGAACCAAGCUCGGCU371 188-206 A-143876 AGCCGAGCUUGGUUCCACU 685 188-206 AD-71939 A-143877CUGUGGAGAGGGGGGCUGA 372 205-223 A-143878 UCAGCCCCCCUCUCCACAG 686 205-223AD-71940 A-143879 CGGGGCUUGCACAGUGAUA 373 223-241 A-143880UAUCACUGUGCAAGCCCCG 687 223-241 AD-71941 A-143881 AUGCUCUCCAAGUAUGAUA374 239-257 A-143882 UAUCAUACUUGGAGAGCAU 688 239-257 AD-71942 A-143885UCGUCCACUUUUCUGCCAA 375 273-291 A-143886 UUGGCAGAAAAGUGGACGA 689 273-291AD-71943 A-143887 AUGCCUGCCUGGCCCCCAU 376 291-309 A-143888AUGGGGGCCAGGCAGGCAU 690 291-309 AD-71944 A-143889 AUCUGCUCCUUGCACCAUA377 308-326 A-143890 UAUGGUGCAAGGAGCAGAU 691 308-326 AD-71945 A-143891UGUUGCAGUGACAACUGUA 378 325-343 A-143892 UACAGUUGUCACUGCAACA 692 325-343AD-71946 A-143893 UGGAAGGAAUAGGAAGCAA 379 342-360 A-143894UUGCUUCCUAUUCCUUCCA 693 342-360 AD-71947 A-143895 CACCAAGACGAGGCUGCAU380 358-376 A-143896 AUGCAGCCUCGUCUUGGUG 694 358-376 AD-71948 A-143897UCCUGUGCAGGAGAGAAUU 381 376-394 A-143898 AAUUCUCUCCUGCACAGGA 695 376-394AD-71949 A-143899 AUUGCCAAAAGCCACGGCU 382 392-410 A-143900AGCCGUGGCUUUUGGCAAU 696 392-410 AD-71950 A-143901 UCCCAGUGCGGGUUCUGCA383 410-428 A-143902 UGCAGAACCCGCACUGGGA 697 410-428 AD-71951 A-143903UGCACCCCUGGCAUCGUCA 384 425-443 A-143904 UGACGAUGCCAGGGGUGCA 698 425-443AD-71952 A-143905 UGAGUAUGUACACACUGCU 385 444-462 A-143906AGCAGUGUGUACAUACUCA 699 444-462 AD-71953 A-143907 UGCUCCGGAAUCAGCCCGA386 459-477 A-143908 UCGGGCUGAUUCCGGAGCA 700 459-477 AD-71954 A-143909AGCCCACCAUGGAGGAGAU 387 477-495 A-143910 AUCUCCUCCAUGGUGGGCU 701 477-495AD-71955 A-143911 UUGAGAAUGCCUUCCAAGA 388 495-513 A-143912UCUUGGAAGGCAUUCUCAA 702 495-513 AD-71956 A-143913 AAGGAAAUCUGUGCCGCUA389 510-528 A-143914 UAGCGGCACAGAUUUCCUU 703 510-528 AD-71957 A-143915CACAGGCUACAGACCCAUA 390 529-547 A-143916 UAUGGGUCUGUAGCCUGUG 704 529-547AD-71958 A-143917 CAUCCUCCAGGGCUUCCGA 391 544-562 A-143918UCGGAAGCCCUGGAGGAUG 705 544-562 AD-71959 A-143919 ACCUUUGCCAGGGAUGGUA392 563-581 A-143920 UACCAUCCCUGGCAAAGGU 706 563-581 AD-71960 A-143921UGGAUGCUGUGGAGGAGAU 393 580-598 A-143922 AUCUCCUCCACAGCAUCCA 707 580-598AD-71961 A-143923 GAUGGGAAUAAUCCAAAUU 394 596-614 A-143924AAUUUGGAUUAUUCCCAUC 708 596-614 AD-71962 A-143927 AGAAAGACCACUCAGUCAA395 630-648 A-143928 UUGACUGAGUGGUCUUUCU 709 630-648 AD-71963 A-143929AGCCUCUCGCCAUCUUUAU 396 647-665 A-143930 AUAAAGAUGGCGAGAGGCU 710 647-665AD-71964 A-143931 UAUUCAAACCAGAGGAGUU 397 663-681 A-143932AACUCCUCUGGUUUGAAUA 711 663-681 AD-71965 A-143933 UUCACGCCCCUGGAUCCAA398 680-698 A-143934 UUGGAUCCAGGGGCGUGAA 712 680-698 AD-71966 A-143935AACCCAGGAGCCCAUUUUU 399 697-715 A-143936 AAAAAUGGGCUCCUGGGUU 713 697-715AD-71967 A-143937 UUCCCCCAGAGUUGCUGAA 400 714-732 A-143938UUCAGCAACUCUGGGGGAA 714 714-732 AD-71968 A-143939 AGGCUGAAAGACACUCCUA401 731-749 A-143940 UAGGAGUGUCUUUCAGCCU 715 731-749 AD-71969 A-143941UCGGAAGCAGCUGCGAUUU 402 748-766 A-143942 AAAUCGCAGCUGCUUCCGA 716 748-766AD-71970 A-143943 UUGAAGGGGAGCGUGUGAA 403 765-783 A-143944UUCACACGCUCCCCUUCAA 717 765-783 AD-71971 A-143945 CGUGGAUACAGGCCUCAAA404 783-801 A-143946 UUUGAGGCCUGUAUCCACG 718 783-801 AD-71972 A-143947AACCCUCAAGGAGCUGCUA 405 799-817 A-143948 UAGCAGCUCCUUGAGGGUU 719 799-817AD-71973 A-143949 UGGACCUCAAGGCUCAGCA 406 816-834 A-143950UGCUGAGCCUUGAGGUCCA 720 816-834 AD-71974 A-143951 ACCCUGACGCCAAGCUGGU407 834-852 A-143952 ACCAGCUUGGCGUCAGGGU 721 834-852 AD-71975 A-143953UCGUGGGGAACACGGAGAU 408 852-870 A-143954 AUCUCCGUGUUCCCCACGA 722 852-870AD-71976 A-143955 AUUGGCAUUGAGAUGAAGU 409 869-887 A-143956ACUUCAUCUCAAUGCCAAU 723 869-887 AD-71977 A-143957 AAGUUCAAGAAUAUGCUGU410 884-902 A-143958 ACAGCAUAUUCUUGAACUU 724 884-902 AD-71978 A-143959UUUCCUAUGAUUGUCUGCA 411 902-920 A-143960 UGCAGACAAUCAUAGGAAA 725 902-920AD-71979 A-143961 CCCAGCCUGGAUCCCUGAA 412 919-937 A-143962UUCAGGGAUCCAGGCUGGG 726 919-937 AD-71980 A-143963 AGCUGAAUUCGGUAGAACA413 936-954 A-143964 UGUUCUACCGAAUUCAGCU 727 936-954 AD-71981 A-143965CAUGGACCCGACGGUAUCU 414 953-971 A-143966 AGAUACCGUCGGGUCCAUG 728 953-971AD-71982 A-143967 UCUCCUUUGGAGCUGCUUA 415 969-987 A-143968UAAGCAGCUCCAAAGGAGA 729 969-987 AD-71983 A-143969 UGCCCCCUGAGCAUUGUGA416  986-1004 A-143970 UCACAAUGCUCAGGGGGCA 730  986-1004 AD-71984A-143971 AAAAAACCCUGGUGGAUGA 417 1005-1023 A-143972 UCAUCCACCAGGGUUUUUU731 1005-1023 AD-71985 A-143973 UGCUGUUGCUAAGCUUCCU 418 1021-1039A-143974 AGGAAGCUUAGCAACAGCA 732 1021-1039 AD-71986 A-143975CCUGCCCAAAAGACAGAGA 419 1037-1055 A-143976 UCUCUGUCUUUUGGGCAGG 7331037-1055 AD-71987 A-143977 UGUUCAGAGGGGUCCUGGA 420 1056-1074 A-143978UCCAGGACCCCUCUGAACA 734 1056-1074 AD-71988 A-143979 UGGAGCAGCUGCGCUGGUU421 1071-1089 A-143980 AACCAGCGCAGCUGCUCCA 735 1071-1089 AD-71989A-143981 UUUGCUGGGAAGCAAGUCA 422 1088-1106 A-143982 UGACUUGCUUCCCAGCAAA736 1088-1106 AD-71990 A-143983 CAAGUCUGUGGCGUCCGUU 423 1105-1123A-143984 AACGGACGCCACAGACUUG 737 1105-1123 AD-71991 A-143985UGGAGGGAACAUCAUCACU 424 1123-1141 A-143986 AGUGAUGAUGUUCCCUCCA 7381123-1141 AD-71992 A-143987 UGCCAGCCCCAUCUCCGAA 425 1141-1159 A-143988UUCGGAGAUGGGGCUGGCA 739 1141-1159 AD-71993 A-143989 ACCUCAACCCCGUGUUCAU426 1158-1176 A-143990 AUGAACACGGGGUUGAGGU 740 1158-1176 AD-71994A-143991 UCAUGGCCAGUGGGGCCAA 427 1173-1191 A-143992 UUGGCCCCACUGGCCAUGA741 1173-1191 AD-71995 A-143993 AAGCUGACACUUGUGUCCA 428 1190-1208A-143994 UGGACACAAGUGUCAGCUU 742 1190-1208 AD-71996 A-143995CAGAGGCACCAGGAGAACU 429 1207-1225 A-143996 AGUUCUCCUGGUGCCUCUG 7431207-1225 AD-71997 A-143997 UGUCCAGAUGGACCACACA 430 1225-1243 A-143998UGUGUGGUCCAUCUGGACA 744 1225-1243 AD-71998 A-143999 CCUUCUUCCCUGGCUACAA431 1242-1260 A-144000 UUGUAGCCAGGGAAGAAGG 745 1242-1260 AD-71999A-144001 AGAAAGACCCUGCUGAGCA 432 1259-1277 A-144002 UGCUCAGCAGGGUCUUUCU746 1259-1277 AD-72000 A-144003 CCGGAGGAGAUACUGCUCU 433 1277-1295A-144004 AGAGCAGUAUCUCCUCCGG 747 1277-1295 AD-72001 A-144005CUCUCCAUAGAGAUCCCCU 434 1292-1310 A-144006 AGGGGAUCUCUAUGGAGAG 7481292-1310 AD-72002 A-144007 UACAGCAGGGAGGGGGAGU 435 1310-1328 A-144008ACUCCCCCUCCCUGCUGUA 749 1310-1328 AD-72003 A-144009 AGUAUUUCUCAGCAUUCAA436 1326-1344 A-144010 UUGAAUGCUGAGAAAUACU 750 1326-1344 AD-72004A-144011 AAGCAGGCCUCCCGGAGAA 437 1343-1361 A-144012 UUCUCCGGGAGGCCUGCUU751 1343-1361 AD-72005 A-144013 AAGAUGACAUUGCCAAGGU 438 1362-1380A-144014 ACCUUGGCAAUGUCAUCUU 752 1362-1380 AD-72006 A-144015GGUAACCAGUGGCAUGAGA 439 1378-1396 A-144016 UCUCAUGCCACUGGUUACC 7531378-1396 AD-72007 A-144017 AGAGUUUUAUUCAAGCCAA 440 1394-1412 A-144018UUGGCUUGAAUAAAACUCU 754 1394-1412 AD-72008 A-144019 AGGAACCACAGAGGUACAA441 1411-1429 A-144020 UUGUACCUCUGUGGUUCCU 755 1411-1429 AD-72009A-144021 AGGAGCUGGCCCUUUGCUA 442 1428-1446 A-144022 UAGCAAAGGGCCAGCUCCU756 1428-1446 AD-72010 A-144023 UAUGGUGGAAUGGCCAACA 443 1445-1463A-144024 UGUUGGCCAUUCCACCAUA 757 1445-1463 AD-72011 A-144025AGAACCAUCUCAGCCCUCA 444 1463-1481 A-144026 UGAGGGCUGAGAUGGUUCU 7581463-1481 AD-72012 A-144027 AAGACCACUCAGAGGCAGA 445 1481-1499 A-144028UCUGCCUCUGAGUGGUCUU 759 1481-1499 AD-72013 A-144029 CAGCUUUCCAAGCUCUGGA446 1496-1514 A-144030 UCCAGAGCUUGGAAAGCUG 760 1496-1514 AD-72014A-144031 AGGAGGAGCUGCUGCAGGA 447 1515-1533 A-144032 UCCUGCAGCAGCUCCUCCU761 1515-1533 AD-72015 A-144033 AGGACGUGUGUGCAGGACU 448 1530-1548A-144034 AGUCCUGCACACACGUCCU 762 1530-1548 AD-72016 A-144035UGGCAGAGGAGCUGCAUCU 449 1548-1566 A-144036 AGAUGCAGCUCCUCUGCCA 7631548-1566 AD-72017 A-144037 UCUGCCUCCCGAUGCCCCU 450 1564-1582 A-144038AGGGGCAUCGGGAGGCAGA 764 1564-1582 AD-72018 A-144039 GGUGGCAUGGUGGACUUCA451 1583-1601 A-144040 UGAAGUCCACCAUGCCACC 765 1583-1601 AD-72019A-144041 UUCCGGUGCACCCUCACCA 452 1598-1616 A-144042 UGGUGAGGGUGCACCGGAA766 1598-1616 AD-71752 A-144043 UCAGCUUCUUCUUCAAGUU 453 1617-1635A-144044 AACUUGAAGAAGAAGCUGA 767 1617-1635 AD-71753 A-144045UUCUACCUGACAGUCCUUA 454 1634-1652 A-144046 UAAGGACUGUCAGGUAGAA 7681634-1652 AD-71754 A-144049 GAGAACCUGGAAGACAAGU 455 1667-1685 A-144050ACUUGUCUUCCAGGUUCUC 769 1667-1685 AD-71755 A-144051 UGUGGUAAACUGGACCCCA456 1685-1703 A-144052 UGGGGUCCAGUUUACCACA 770 1685-1703 AD-71756A-144053 CACUUUCGCCAGUGCAACU 457 1702-1720 A-144054 AGUUGCACUGGCGAAAGUG771 1702-1720 AD-71757 A-144055 ACUUUACUGUUUCAGAAAG 458 1718-1736A-144056 CUUUCUGAAACAGUAAAGU 772 1718-1736 AD-71758 A-144057AAGACCCCCCAGCCGAUGU 459 1734-1752 A-144058 ACAUCGGCUGGGGGGUCUU 7731734-1752 AD-71759 A-144059 UCCAGCUCUUCCAAGAGGU 460 1752-1770 A-144060ACCUCUUGGAAGAGCUGGA 774 1752-1770 AD-71760 A-144061 UGCCCAAGGGUCAGUCUGA461 1770-1788 A-144062 UCAGACUGACCCUUGGGCA 775 1770-1788 AD-71761A-144063 GAGGAGGACAUGGUGGGCA 462 1787-1805 A-144064 UGCCCACCAUGUCCUCCUC776 1787-1805 AD-71762 A-144065 GCCGGCCCCUGCCCCACCU 463 1803-1821A-144066 AGGUGGGGCAGGGGCCGGC 777 1803-1821 AD-71763 A-144067CCUGGCAGCGGACAUGCAA 464 1819-1837 A-144068 UUGCAUGUCCGCUGCCAGG 7781819-1837 AD-71764 A-144069 AGGCCUCUGGUGAGGCCGU 465 1836-1854 A-144070ACGGCCUCACCAGAGGCCU 779 1836-1854 AD-71765 A-144071 UGUACUGUGACGACAUUCA466 1854-1872 A-144072 UGAAUGUCGUCACAGUACA 780 1854-1872 AD-71766A-144073 CUCGCUACGAGAAUGAGCU 467 1872-1890 A-144074 AGCUCAUUCUCGUAGCGAG781 1872-1890 AD-71767 A-144075 CUGUCUCUCCGGCUGGUCA 468 1889-1907A-144076 UGACCAGCCGGAGAGACAG 782 1889-1907 AD-71768 A-144079CCACGCCAAGAUCAAGUCA 469 1921-1939 A-144080 UGACUUGAUCUUGGCGUGG 7831921-1939 AD-71769 A-144081 CAUAGAUACAUCAGAAGCU 470 1939-1957 A-144082AGCUUCUGAUGUAUCUAUG 784 1939-1957 AD-71770 A-144085 UUUGUUUGUUUCAUUUCCA471 1973-1991 A-144086 UGGAAAUGAAACAAACAAA 785 1973-1991 AD-71771A-144087 GCUGAUGAUGUUCCUGGGA 472 1991-2009 A-144088 UCCCAGGAACAUCAUCAGC786 1991-2009 AD-71772 A-144093 CAGUCUUUGCGAAGGAUAA 473 2040-2058A-144094 UUAUCCUUCGCAAAGACUG 787 2040-2058 AD-71773 A-144095AAGGUUACUUGUGUUGGGA 474 2057-2075 A-144096 UCCCAACACAAGUAACCUU 7882057-2075 AD-71774 A-144097 CAUAUCAUUGGUGCUGUGA 475 2075-2093 A-144098UCACAGCACCAAUGAUAUG 789 2075-2093 AD-71775 A-144099 UGGUUGCUGACACCCCGGA476 2091-2109 A-144100 UCCGGGGUGUCAGCAACCA 790 2091-2109 AD-71776A-144101 ACACACACAGAGAGCUGCA 477 2110-2128 A-144102 UGCAGCUCUCUGUGUGUGU791 2110-2128 AD-71777 A-144103 GCCCAAGGGGUGAAAAUCA 478 2126-2144A-144104 UGAUUUUCACCCCUUGGGC 792 2126-2144 AD-71778 A-144105UCACCUAUGAAGAACUACA 479 2142-2160 A-144106 UGUAGUUCUUCAUAGGUGA 7932142-2160 AD-71779 A-144109 GAGGAUGCUAUAAAGAACA 480 2177-2195 A-144110UGUUCUUUAUAGCAUCCUC 794 2177-2195 AD-71780 A-144111 CAACUCCUUUUAUGGACCU481 2194-2212 A-144112 AGGUCCAUAAAAGGAGUUG 795 2194-2212 AD-71781A-144113 CCUGAGCUGAAGAUCGAGA 482 2210-2228 A-144114 UCUCGAUCUUCAGCUCAGG796 2210-2228 AD-71782 A-144115 AAAGGGGACCUAAAGAAGA 483 2228-2246A-144116 UCUUCUUUAGGUCCCCUUU 797 2228-2246 AD-71783 A-144117AGGGGUUUUCCGAAGCAGA 484 2244-2262 A-144118 UCUGCUUCGGAAAACCCCU 7982244-2262 AD-71784 A-144119 UAAUGUUGUGUCAGGGGAA 485 2263-2281 A-144120UUCCCCUGACACAACAUUA 799 2263-2281 AD-71785 A-144121 AGAUAUACAUCGGUGGCCA486 2280-2298 A-144122 UGGCCACCGAUGUAUAUCU 800 2280-2298 AD-71786A-144123 GCCAAGAGCACUUCUACCU 487 2295-2313 A-144124 AGGUAGAAGUGCUCUUGGC801 2295-2313 AD-71787 A-144125 GGAGACUCACUGCACCAUU 488 2314-2332A-144126 AAUGGUGCAGUGAGUCUCC 802 2314-2332 AD-71788 A-144127UUGCUGUUCCAAAAGGCGA 489 2331-2349 A-144128 UCGCCUUUUGGAACAGCAA 8032331-2349 AD-71789 A-144129 CGAGGCAGGGGAGAUGGAA 490 2347-2365 A-144130UUCCAUCUCCCCUGCCUCG 804 2347-2365 AD-71790 A-144131 AGCUCUUUGUGUCUACACA491 2364-2382 A-144132 UGUGUAGACACAAAGAGCU 805 2364-2382 AD-71791A-144133 CAGAACACCAUGAAGACCA 492 2381-2399 A-144134 UGGUCUUCAUGGUGUUCUG806 2381-2399 AD-71792 A-144135 CAGAGCUUUGUUGCAAAAA 493 2399-2417A-144136 UUUUUGCAACAAAGCUCUG 807 2399-2417 AD-71793 A-144137AAAAUGUUGGGGGUUCCAA 494 2414-2432 A-144138 UUGGAACCCCCAACAUUUU 8082414-2432 AD-71794 A-144139 AGCAAACCGGAUUGUGGUU 495 2431-2449 A-144140AACCACAAUCCGGUUUGCU 809 2431-2449 AD-71795 A-144141 UUCGAGUGAAGAGAAUGGA496 2448-2466 A-144142 UCCAUUCUCUUCACUCGAA 810 2448-2466 AD-71796A-144143 AGGAGGCUUUGGAGGCAAA 497 2467-2485 A-144144 UUUGCCUCCAAAGCCUCCU811 2467-2485 AD-71797 A-144145 AAGGAGACCCGGAGCACUA 498 2483-2501A-144146 UAGUGCUCCGGGUCUCCUU 812 2483-2501 AD-71798 A-144147UGUGGUGUCCACGGCAGUA 499 2500-2518 A-144148 UACUGCCGUGGACACCACA 8132500-2518 AD-71799 A-144149 UGGCCCUGGCUGCAUAUAA 500 2517-2535 A-144150UUAUAUGCAGCCAGGGCCA 814 2517-2535 AD-71800 A-144153 UGCGAUGCAUGCUGGACCA501 2550-2568 A-144154 UGGUCCAGCAUGCAUCGCA 815 2550-2568 AD-71801A-144155 CGUGAUGAGGACAUGCUGA 502 2567-2585 A-144156 UCAGCAUGUCCUCAUCACG816 2567-2585 AD-71802 A-144157 UAACUGGUGGCAGACAUCA 503 2586-2604A-144158 UGAUGUCUGCCACCAGUUA 817 2586-2604 AD-71803 A-144159UCCCUUCCUGGCCAGAUAA 504 2602-2620 A-144160 UUAUCUGGCCAGGAAGGGA 8182602-2620 AD-71804 A-144161 UACAAGGUUGGCUUCAUGA 505 2618-2636 A-144162UCAUGAAGCCAACCUUGUA 819 2618-2636 AD-71805 A-144163 AGACUGGGACAGUUGUGGA506 2637-2655 A-144164 UCCACAACUGUCCCAGUCU 820 2637-2655 AD-71806A-144165 GCUCUUGAGGUGGACCACU 507 2654-2672 A-144166 AGUGGUCCACCUCAAGAGC821 2654-2672 AD-71807 A-144167 ACUUCAGCAAUGUGGGGAA 508 2670-2688A-144168 UUCCCCACAUUGCUGAAGU 822 2670-2688 AD-71808 A-144169GAACACCCAGGAUCUCUCU 509 2686-2704 A-144170 AGAGAGAUCCUGGGUGUUC 8232686-2704 AD-71809 A-144171 CAGAGUAUUAUGGAACGAA 510 2705-2723 A-144172UUCGUUCCAUAAUACUCUG 824 2705-2723 AD-71810 A-144173 AGCUUUAUUCCACAUGGAA511 2722-2740 A-144174 UUCCAUGUGGAAUAAAGCU 825 2722-2740 AD-71811A-144175 ACAACUGCUAUAAAAUCCA 512 2739-2757 A-144176 UGGAUUUUAUAGCAGUUGU826 2739-2757 AD-71812 A-144177 UCCCCAACAUCCGGGGCAA 513 2754-2772A-144178 UUGCCCCGGAUGUUGGGGA 827 2754-2772 AD-71813 A-144179UGGGCGGCUGUGCAAAACA 514 2773-2791 A-144180 UGUUUUGCACAGCCGCCCA 8282773-2791 AD-71814 A-144181 AACCAACCUUCCCUCCAAA 515 2788-2806 A-144182UUUGGAGGGAAGGUUGGUU 829 2788-2806 AD-71815 A-144183 ACACGGCCUUCCGGGGCUU516 2805-2823 A-144184 AAGCCCCGGAAGGCCGUGU 830 2805-2823 AD-71816A-144185 UUGGGGGGCCCCAGGGGAU 517 2823-2841 A-144186 AUCCCCUGGGGCCCCCCAA831 2823-2841 AD-71817 A-144187 AUGCUCAUUGCCGAGUGCU 518 2840-2858A-144188 AGCACUCGGCAAUGAGCAU 832 2840-2858 AD-71818 A-144189UGGAUGAGUGAAGUUGCAA 519 2858-2876 A-144190 UUGCAACUUCACUCAUCCA 8332858-2876 AD-71819 A-144191 AGUGACCUGUGGGAUGCCU 520 2875-2893 A-144192AGGCAUCCCACAGGUCACU 834 2875-2893 AD-71820 A-144193 CCUGCAGAGGAGGUGCGGA521 2891-2909 A-144194 UCCGCACCUCCUCUGCAGG 835 2891-2909 AD-71821A-144195 AGAAAAAACCUGUACAAAG 522 2909-2927 A-144196 CUUUGUACAGGUUUUUUCU836 2909-2927 AD-71822 A-144197 AAAGAAGGGGACCUGACAA 523 2924-2942A-144198 UUGUCAGGUCCCCUUCUUU 837 2924-2942 AD-71823 A-144199ACACUUCAACCAGAAGCUU 524 2941-2959 A-144200 AAGCUUCUGGUUGAAGUGU 8382941-2959 AD-71824 A-144201 UUGAGGGUUUCACCUUGCA 525 2958-2976 A-144202UGCAAGGUGAAACCCUCAA 839 2958-2976 AD-71825 A-144203 CAGAUGCUGGGAAGAAUGA526 2977-2995 A-144204 UCAUUCUUCCCAGCAUCUG 840 2977-2995 AD-71826A-144205 UGCCUAGCAAGCUCUCAGU 527 2993-3011 A-144206 ACUGAGAGCUUGCUAGGCA841 2993-3011 AD-71827 A-144207 UAUCAUGCUCGGAAGAGUA 528 3011-3029A-144208 UACUCUUCCGAGCAUGAUA 842 3011-3029 AD-71828 A-144209AGUGAGGUUGACAAGUUCA 529 3026-3044 A-144210 UGAACUUGUCAACCUCACU 8433026-3044 AD-71829 A-144211 AACAAGGAGAAUUGUUGGA 530 3044-3062 A-144212UCCAACAAUUCUCCUUGUU 844 3044-3062 AD-71830 A-144213 AAAAAGAGAGGAUUGUGCA531 3062-3080 A-144214 UGCACAAUCCUCUCUUUUU 845 3062-3080 AD-71831A-144215 CAUAAUUCCCACCAAGUUU 532 3079-3097 A-144216 AAACUUGGUGGGAAUUAUG846 3079-3097 AD-71832 A-144217 UUUGGAAUAAGCUUUACAA 533 3095-3113A-144218 UUGUAAAGCUUAUUCCAAA 847 3095-3113 AD-71833 A-144219AGUUCCUUUUCUGAAUCAA 534 3112-3130 A-144220 UUGAUUCAGAAAAGGAACU 8483112-3130 AD-71834 A-144221 AGGCAGGAGCCCUACUUCA 535 3129-3147 A-144222UGAAGUAGGGCUCCUGCCU 849 3129-3147 AD-71835 A-144223 CAUGUGUACACAGAUGGCU536 3146-3164 A-144224 AGCCAUCUGUGUACACAUG 850 3146-3164 AD-71836A-144225 UCUGUGCUGCUGACCCACA 537 3164-3182 A-144226 UGUGGGUCAGCAGCACAGA851 3164-3182 AD-71837 A-144229 GGCCAAGGCCUUCAUACCA 538 3197-3215A-144230 UGGUAUGAAGGCCUUGGCC 852 3197-3215 AD-71838 A-144231AAAAUGGUCCAGGUGGCCA 539 3215-3233 A-144232 UGGCCACCUGGACCAUUUU 8533215-3233 AD-71839 A-144233 GCCAGUAGAGCUCUGAAAA 540 3230-3248 A-144234UUUUCAGAGCUCUACUGGC 854 3230-3248 AD-71840 A-144235 AAUCCCCACCUCUAAGAUU541 3247-3265 A-144236 AAUCUUAGAGGUGGGGAUU 855 3247-3265 AD-71841A-144237 UAUAUCAGCGAGACAAGCA 542 3266-3284 A-144238 UGCUUGUCUCGCUGAUAUA856 3266-3284 AD-71842 A-144239 AGCACUAACACUGUGCCCA 543 3281-3299A-144240 UGGGCACAGUGUUAGUGCU 857 3281-3299 AD-71843 A-144241CAACACCUCUCCCACGGCU 544 3298-3316 A-144242 AGCCGUGGGAGAGGUGUUG 8583298-3316 AD-71844 A-144243 UGCCUCUGUCAGCGCUGAA 545 3316-3334 A-144244UUCAGCGCUGACAGAGGCA 859 3316-3334 AD-71845 A-144245 ACCUCAAUGGACAGGCCGU546 3333-3351 A-144246 ACGGCCUGUCCAUUGAGGU 860 3333-3351 AD-71846A-144247 GUCUAUGCGGCUUGUCAGA 547 3350-3368 A-144248 UCUGACAAGCCGCAUAGAC861 3350-3368 AD-71847 A-144249 AGACCAUCUUGAAAAGGCU 548 3366-3384A-144250 AGCCUUUUCAAGAUGGUCU 862 3366-3384 AD-71848 A-144251UGGAACCCUACAAGAAGAA 549 3384-3402 A-144252 UUCUUCUUGUAGGGUUCCA 8633384-3402 AD-71849 A-144253 AAGAAUCCCAGUGGCUCCU 550 3401-3419 A-144254AGGAGCCACUGGGAUUCUU 864 3401-3419 AD-71850 A-144255 UGGGAAGACUGGGUCACAA551 3419-3437 A-144256 UUGUGACCCAGUCUUCCCA 865 3419-3437 AD-71851A-144257 ACAGCUGCCUACAUGGACA 552 3434-3452 A-144258 UGUCCAUGUAGGCAGCUGU866 3434-3452 AD-71852 A-144259 ACAGUGAGCUUGUCUGCCA 553 3452-3470A-144260 UGGCAGACAAGCUCACUGU 867 3452-3470 AD-71853 A-144261ACUGGGUUUUAUAGAACAA 554 3470-3488 A-144262 UUGUUCUAUAAAACCCAGU 8683470-3488 AD-71854 A-144263 ACCCAAUCUGGGCUACAGA 555 3487-3505 A-144264UCUGUAGCCCAGAUUGGGU 869 3487-3505 AD-71855 A-144265 CAGCUUUGAGACUAACUCA556 3502-3520 A-144266 UGAGUUAGUCUCAAAGCUG 870 3502-3520 AD-71856A-144267 GGGAACCCCUUCCACUACU 557 3521-3539 A-144268 AGUAGUGGAAGGGGUUCCC871 3521-3539 AD-71857 A-144269 CUUCAGCUAUGGGGUGGCU 558 3538-3556A-144270 AGCCACCCCAUAGCUGAAG 872 3538-3556 AD-71858 A-144271CUUGCUCUGAAGUAGAAAU 559 3555-3573 A-144272 AUUUCUACUUCAGAGCAAG 8733555-3573 AD-71859 A-144273 AAAUCGACUGCCUAACAGA 560 3570-3588 A-144274UCUGUUAGGCAGUCGAUUU 874 3570-3588 AD-71860 A-144275 GAGAUCAUAAGAACCUCCA561 3588-3606 A-144276 UGGAGGUUCUUAUGAUCUC 875 3588-3606 AD-71861A-144277 GCACAGAUAUUGUCAUGGA 562 3606-3624 A-144278 UCCAUGACAAUAUCUGUGC876 3606-3624 AD-71862 A-144279 UGGAUGUUGGCUCCAGUCU 563 3621-3639A-144280 AGACUGGAGCCAACAUCCA 877 3621-3639 AD-71863 A-144281UAAACCCUGCCAUUGAUAU 564 3639-3657 A-144282 AUAUCAAUGGCAGGGUUUA 8783639-3657 AD-71864 A-144283 UUGGACAGGUGGAAGGGGA 565 3657-3675 A-144284UCCCCUUCCACCUGUCCAA 879 3657-3675 AD-71865 A-144285 GGGCAUUUGUCCAGGGCCU566 3672-3690 A-144286 AGGCCCUGGACAAAUGCCC 880 3672-3690 AD-71866A-144287 UUGGCCUCUUCACCCUAGA 567 3690-3708 A-144288 UCUAGGGUGAAGAGGCCAA881 3690-3708 AD-71867 A-144289 AGAGGAGCUACACUAUUCA 568 3706-3724A-144290 UGAAUAGUGUAGCUCCUCU 882 3706-3724 AD-71868 A-144291CCCCGAGGGGAGCCUGCAA 569 3724-3742 A-144292 UUGCAGGCUCCCCUCGGGG 8833724-3742 AD-71869 A-144293 CACACCCGUGGCCCUAGCA 570 3740-3758 A-144294UGCUAGGGCCACGGGUGUG 884 3740-3758 AD-71870 A-144295 ACCUACAAGAUCCCGGCAU571 3758-3776 A-144296 AUGCCGGGAUCUUGUAGGU 885 3758-3776 AD-71871A-144297 UUUGGCAGCAUCCCCAUUA 572 3776-3794 A-144298 UAAUGGGGAUGCUGCCAAA886 3776-3794 AD-71872 A-144301 CCUGCUCCGCGACUGCCCA 573 3808-3826A-144302 UGGGCAGUCGCGGAGCAGG 887 3808-3826 AD-71873 A-144303CAACAAGAAGGCCAUCUAU 574 3826-3844 A-144304 AUAGAUGGCCUUCUUGUUG 8883826-3844 AD-71874 A-144305 UAUGCAUCGAAGGCUGUUA 575 3842-3860 A-144306UAACAGCCUUCGAUGCAUA 889 3842-3860 AD-71875 A-144307 UGGAGAGCCGCCCCUCUUA576 3859-3877 A-144308 UAAGAGGGGCGGCUCUCCA 890 3859-3877 AD-71876A-144309 UCCUGGCUGCUUCUAUCUU 577 3876-3894 A-144310 AAGAUAGAAGCAGCCAGGA891 3876-3894 AD-71877 A-144311 UUCUUUGCCAUCAAAGAUA 578 3893-3911A-144312 UAUCUUUGAUGGCAAAGAA 892 3893-3911 AD-71878 A-144313CCAUCCGUGCAGCUCGAGA 579 3912-3930 A-144314 UCUCGAGCUGCACGGAUGG 8933912-3930 AD-71879 A-144315 AGCUCAGCACACAGGUAAU 580 3928-3946 A-144316AUUACCUGUGUGCUGAGCU 894 3928-3946 AD-71880 A-144317 AAUAACGUGAAGGAACUCU581 3944-3962 A-144318 AGAGUUCCUUCACGUUAUU 895 3944-3962 AD-71881A-144319 UUCCGGCUAGACAGCCCUA 582 3962-3980 A-144320 UAGGGCUGUCUAGCCGGAA896 3962-3980 AD-71882 A-144321 UGCCACCCCGGAGAAGAUA 583 3979-3997A-144322 UAUCUUCUCCGGGGUGGCA 897 3979-3997 AD-71883 A-144323AUCCGCAAUGCCUGCGUGA 584 3995-4013 A-144324 UCACGCAGGCAUUGCGGAU 8983995-4013 AD-71884 A-144325 GACAAGUUCACCACCCUGU 585 4013-4031 A-144326ACAGGGUGGUGAACUUGUC 899 4013-4031 AD-71885 A-144327 UGUGUGUCACUGGUGUCCA586 4029-4047 A-144328 UGGACACCAGUGACACACA 900 4029-4047 AD-71886A-144329 AGAAAACUGCAAACCCUGA 587 4048-4066 A-144330 UCAGGGUUUGCAGUUUUCU901 4048-4066 AD-71887 A-144331 CUGGUCUGUGAGGGUCUAA 588 4063-4081A-144332 UUAGACCCUCACAGACCAG 902 4063-4081 AD-71888 A-144333AAAGAGAGAGUCCUCAGCA 589 4080-4098 A-144334 UGCUGAGGACUCUCUCUUU 9034080-4098 AD-71889 A-144335 AGAGUCUUCUUGUGCUGCA 590 4098-4116 A-144336UGCAGCACAAGAAGACUCU 904 4098-4116 AD-71890 A-144337 GCCUUUGGGCUUCCAUGGA591 4114-4132 A-144338 UCCAUGGAAGCCCAAAGGC 905 4114-4132 AD-71891A-144341 CAGAACAUGGAUCUAUUAA 592 4150-4168 A-144342 UUAAUAGAUCCAUGUUCUG906 4150-4168 AD-71892 A-144343 UUAAAGUCACAGAAUGACA 593 4165-4183A-144344 UGUCAUUCUGUGACUUUAA 907 4165-4183 AD-71893 A-144345AGACCUGUGAUUUGUCAAA 594 4183-4201 A-144346 UUUGACAAAUCACAGGUCU 9084183-4201 AD-71894 A-144347 AGAUGGGAUUUGGAAGACA 595 4200-4218 A-144348UGUCUUCCAAAUCCCAUCU 909 4200-4218 AD-71895 A-144349 AAGUGAAUGCAAUGGAAGA596 4218-4236 A-144350 UCUUCCAUUGCAUUCACUU 910 4218-4236 AD-71896A-144351 AAGAUUUUGAUCAAAAAUA 597 4233-4251 A-144352 UAUUUUUGAUCAAAAUCUU911 4233-4251 AD-71897 A-144353 UGUAAUUUGUAAACACAAU 598 4250-4268A-144354 AUUGUGUUUACAAAUUACA 912 4250-4268 AD-71898 A-144355GAUAAGCAAAUUCAAAACU 599 4269-4287 A-144356 AGUUUUGAAUUUGCUUAUC 9134269-4287 AD-71899 A-144357 ACUGUUAUGCCUAAAUGGU 600 4285-4303 A-144358ACCAUUUAGGCAUAACAGU 914 4285-4303 AD-71900 A-144359 UGAAUAUGCAAUUAGGAUA601 4303-4321 A-144360 UAUCCUAAUUGCAUAUUCA 915 4303-4321 AD-71901A-144361 AUCAUUUUCUGUCUGUUUU 602 4319-4337 A-144362 AAAACAGACAGAAAAUGAU916 4319-4337 AD-71902 A-144363 UUAAUCAUGUAUCUGGAAU 603 4336-4354A-144364 AUUCCAGAUACAUGAUUAA 917 4336-4354 AD-71903 A-144365AAUAGGGUCGGGAAGGGUU 604 4352-4370 A-144366 AACCCUUCCCGACCCUAUU 9184352-4370 AD-71904 A-144367 UUUGUGCUAUUCCCCACUU 605 4369-4387 A-144368AAGUGGGGAAUAGCACAAA 919 4369-4387 AD-71905 A-144369 UUACUGGACAGCCUGUAUA606 4386-4404 A-144370 UAUACAGGCUGUCCAGUAA 920 4386-4404 AD-71906A-144371 AACCUCAAGUUCUGAUGGU 607 4404-4422 A-144372 ACCAUCAGAACUUGAGGUU921 4404-4422 AD-71907 A-144373 UGUCUGUCCUUUGAAGAGA 608 4422-4440A-144374 UCUCUUCAAAGGACAGACA 922 4422-4440 AD-71908 A-144375AGGAUUCCCACAAACCUCU 609 4438-4456 A-144376 AGAGGUUUGUGGGAAUCCU 9234438-4456 AD-71909 A-144377 UAGAAGCUUAAACCGAAGU 610 4456-4474 A-144378ACUUCGGUUUAAGCUUCUA 924 4456-4474 AD-71910 A-144379 AAGUUACUUUAAAUCGUGU611 4471-4489 A-144380 ACACGAUUUAAAGUAACUU 925 4471-4489 AD-71911A-144381 UGCCUUCCUGUGAAAGCCU 612 4489-4507 A-144382 AGGCUUUCACAGGAAGGCA926 4489-4507 AD-71912 A-144383 CUGGCCUUCAAACCAAUGA 613 4506-4524A-144384 UCAUUGGUUUGAAGGCCAG 927 4506-4524 AD-71913 A-144385AACAGCAAAGCAUAACCUU 614 4524-4542 A-144386 AAGGUUAUGCUUUGCUGUU 9284524-4542 AD-71914 A-144387 UUGAAUCUAUACUCAAAUU 615 4541-4559 A-144388AAUUUGAGUAUAGAUUCAA 929 4541-4559 AD-71915 A-144389 UUUUGCAAUGAGGCAGUGA616 4558-4576 A-144390 UCACUGCCUCAUUGCAAAA 930 4558-4576 AD-71916A-144391 UGGGGUAAGGUUAAAUCCU 617 4574-4592 A-144392 AGGAUUUAACCUUACCCCA931 4574-4592 AD-71917 A-144393 UCUAACCAUCUUUGAAUCA 618 4592-4610A-144394 UGAUUCAAAGAUGGUUAGA 932 4592-4610 AD-71918 A-144395AUCAUUGGAAAGAAUAAAG 619 4607-4625 A-144396 CUUUAUUCUUUCCAAUGAU 9334607-4625 AD-71919 A-144397 AAUGAAACAAAUUCAAGGU 620 4626-4644 A-144398ACCUUGAAUUUGUUUCAUU 934 4626-4644 AD-71920 A-144399 AGGUUAAUUGGAUCUGAUU621 4641-4659 A-144400 AAUCAGAUCCAAUUAACCU 935 4641-4659 AD-71921A-144401 UUUGUGAAGCUGCAUAAAG 622 4659-4677 A-144402 CUUUAUGCAGCUUCACAAA936 4659-4677 AD-71922 A-144403 AGCAAGAUUACUCUAUAAU 623 4676-4694A-144404 AUUAUAGAGUAAUCUUGCU 937 4676-4694 AD-71923 A-144405UACAAAAAUCCAACCAACU 624 4694-4712 A-144406 AGUUGGUUGGAUUUUUGUA 9384694-4712 AD-71924 A-144407 ACUCAAUUAUUGAGCACGU 625 4710-4728 A-144408ACGUGCUCAAUAAUUGAGU 939 4710-4728 AD-71925 A-144409 UACAAUGUUCUAGAUUUCU626 4728-4746 A-144410 AGAAAUCUAGAACAUUGUA 940 4728-4746 AD-71926A-144411 UUCUUUCCCUUCCUCUUUA 627 4743-4761 A-144412 UAAAGAGGAAGGGAAAGAA941 4743-4761 AD-71927 A-144413 GAAGAGAAUAUUUGUAUUA 628 4761-4779A-144414 UAAUACAAAUAUUCUCUUC 942 4761-4779 AD-71928 A-144415UUCCAAAUACUCUUUGAGU 629 4777-4795 A-144416 ACUCAAAGAGUAUUUGGAA 9434777-4795 AD-71929 A-144417 UAUUUACAAAAAAGAUUAU 630 4795-4813 A-144418AUAAUCUUUUUUGUAAAUA 944 4795-4813 AD-72020 A-144419 UAUGUUUAAUCUUUACAUU631 4811-4829 A-144420 AAUGUAAAGAUUAAACAUA 945 4811-4829 AD-72021A-144421 UUUGAAGCCAAAGUAAUUU 632 4828-4846 A-144422 AAAUUACUUUGGCUUCAAA946 4828-4846 AD-72022 A-144423 UUCCACCUAGAAAUGAUGA 633 4845-4863A-144424 UCAUCAUUUCUAGGUGGAA 947 4845-4863 AD-72023 A-144425UAUCAGUCCUGGCAUGGUA 634 4864-4882 A-144426 UACCAUGCCAGGACUGAUA 9484864-4882 AD-72024 A-144427 UGGCUCACCCCUAUAAUCA 635 4881-4899 A-144428UGAUUAUAGGGGUGAGCCA 949 4881-4899 AD-72025 A-144429 AUCCCAGCACUUUGGGAGA636 4896-4914 A-144430 UCUCCCAAAGUGCUGGGAU 950 4896-4914 AD-72026A-144431 CUAAGGCAGGAGAAUUGCU 637 4915-4933 A-144432 AGCAAUUCUCCUGCCUUAG951 4915-4933 AD-72027 A-144433 UGCUUGAGCCCAGCAGUUU 638 4930-4948A-144434 AAACUGCUGGGCUCAAGCA 952 4930-4948 AD-72028 A-144435UUGAGACCAGCCUGGGCAA 639 4947-4965 A-144436 UUGCCCAGGCUGGUCUCAA 9534947-4965 AD-72029 A-144437 ACAUAGAGAGCUCCUGUCU 640 4965-4983 A-144438AGACAGGAGCUCUCUAUGU 954 4965-4983 AD-72030 A-144439 UCUUUAAAAAAAAUUUUUU641 4981-4999 A-144440 AAAAAAUUUUUUUUAAAGA 955 4981-4999 AD-72031A-144441 UUAAUUAGUUGGUCUUGAU 642 4999-5017 A-144442 AUCAAGACCAACUAAUUAA956 4999-5017 AD-72032 A-144443 UAGUGCAUGCCUGUAGUCA 643 5017-5035A-144444 UGACUACAGGCAUGCACUA 957 5017-5035 AD-72033 A-144445CCCAACUACUUGAAAGGCU 644 5034-5052 A-144446 AGCCUUUCAAGUAGUUGGG 9585034-5052 AD-72034 A-144447 CUGAGGUGGAGAGAUCAUU 645 5051-5069 A-144448AAUGAUCUCUCCACCUCAG 959 5051-5069 AD-72035 A-144449 UUUGAGCUCAGGAGGUUGA646 5068-5086 A-144450 UCAACCUCCUGAGCUCAAA 960 5068-5086 AD-72036A-144451 UUGAGGCUGCAGUGAGCUA 647 5083-5101 A-144452 UAGCUCACUGCAGCCUCAA961 5083-5101 AD-72037 A-144455 CUCCUGCCUGAGCGACUGA 648 5118-5136A-144456 UCAGUCGCUCAGGCAGGAG 962 5118-5136 AD-72038 A-144457UGAGCAAGAUCUUGUCUCU 649 5134-5152 A-144458 AGAGACAAGAUCUUGCUCA 9635134-5152 AD-72039 A-144459 UGAAGAAAAAAAAAGAAAU 650 5152-5170 A-144460AUUUCUUUUUUUUUCUUCA 964 5152-5170 AD-72040 A-144461 UAAAAAUGCUGCUAUCAAA651 5170-5188 A-144462 UUUGAUAGCAGCAUUUUUA 965 5170-5188 AD-72041A-144463 AAAAUCAAGCCCAACCAGA 652 5186-5204 A-144464 UCUGGUUGGGCUUGAUUUU966 5186-5204 AD-72042 A-144465 AGAGGUAGAAGAGCCAAGA 653 5202-5220A-144466 UCUUGGCUCUUCUACCUCU 967 5202-5220 AD-72043 A-144467AAGCCUGGGUUCUCAUCCU 654 5220-5238 A-144468 AGGAUGAGAACCCAGGCUU 9685220-5238 AD-72044 A-144469 CUAGCUCUGUCUCUUCUGU 655 5237-5255 A-144470ACAGAAGAGACAGAGCUAG 969 5237-5255 AD-72045 A-144473 UUGGACUGUCAAUUCCCCU656 5272-5290 A-144474 AGGGGAAUUGACAGUCCAA 970 5272-5290 AD-72046A-144475 CCUUCCUGUGAUCCAUUUU 657 5288-5306 A-144476 AAAAUGGAUCACAGGAAGG971 5288-5306 AD-72047 A-144481 UCUCACGUCUUCUGCUUUA 658 5339-5357A-144482 UAAAGCAGAAGACGUGAGA 972 5339-5357 AD-72048 A-144485UAUGACCUGAAAACUCCAA 659 5372-5390 A-144486 UUGGAGUUUUCAGGUCAUA 9735372-5390 AD-72049 A-144487 UUACAUAAAGGAUCUGCAA 660 5391-5409 A-144488UUGCAGAUCCUUUAUGUAA 974 5391-5409 AD-72050 A-144489 CAGCUAUCUAAGGCUUGGU661 5407-5425 A-144490 ACCAAGCCUUAGAUAGCUG 975 5407-5425 AD-72051A-144493 AUGAUACCUGGGUCUAAUA 662 5440-5458 A-144494 UAUUAGACCCAGGUAUCAU976 5440-5458 AD-72052 A-144495 AACUCUGCUGAGAUCACCU 663 5459-5477A-144496 AGGUGAUCUCAGCAGAGUU 977 5459-5477 AD-72053 A-144497ACCUCAAGUUUCUGCGGUU 664 5474-5492 A-144498 AACCGCAGAAACUUGAGGU 9785474-5492 AD-72054 A-144499 UUGGUAAAGAGAACAAGGA 665 5491-5509 A-144500UCCUUGUUCUCUUUACCAA 979 5491-5509 AD-72055 A-144501 AAGAACAAACAUCCCUUUU666 5510-5528 A-144502 AAAAGGGAUGUUUGUUCUU 980 5510-5528 AD-72056A-144503 UUUUAUUGCUCCAAAUGGU 667 5525-5543 A-144504 ACCAUUUGGAGCAAUAAAA981 5525-5543 AD-72057 A-144505 GAUUUAAUCCCUACAUGGU 668 5544-5562A-144506 ACCAUGUAGGGAUUAAAUC 982 5544-5562 AD-72058 A-144507GGUGCUGGGUGGACAAUGU 669 5560-5578 A-144508 ACAUUGUCCACCCAGCACC 9835560-5578 AD-72059 A-144509 UGUCACUGUCACAUGCCUU 670 5578-5596 A-144510AAGGCAUGUGACAGUGACA 984 5578-5596 AD-72060 A-144511 UUCACUGUAUAAAUCCAAA671 5595-5613 A-144512 UUUGGAUUUAUACAGUGAA 985 5595-5613 AD-72061A-144513 ACCUUCUGCCAGAGAGAAU 672 5612-5630 A-144514 AUUCUCUCUGGCAGAAGGU986 5612-5630 AD-72062 A-144517 CAUGGAGGGAGGAUAGUGA 673 5644-5662A-144518 UCACUAUCCUCCCUCCAUG 987 5644-5662 AD-72063 A-144519AAAUGAUAUAGUUGGACUA 674 5663-5681 A-144520 UAGUCCAACUAUAUCAUUU 9885663-5681 AD-72064 A-144521 ACUGGUGCUUGAUGUCACU 675 5678-5696 A-144522AGUGACAUCAAGCACCAGU 989 5678-5696 AD-72065 A-144523 CUAAUAAAUGAAACUGUCA676 5695-5713 A-144524 UGACAGUUUCAUUUAUUAG 990 5695-5713

TABLE 7 XDH Modified Sequences Anti- Sense SEQ sense SEQ SEQ DuplexOligo ID Oligo ID mRNA ID Name Name Sense OligoSeq NO NameAntisense OligoSeq NO target sequence NO AD-71930 A-143855ACUACCUGCCAGUGUCUCUdTdT  991 A- AGAGACACUGGCAGGUAGUdTdT 1305ACUACCUGCCAGUGUCUCU 1619 143856 AD-71931 A-143857UUAGGAGUGAGGUACCUGAdTdT  992 A- UCAGGUACCUCACUCCUAAdTdT 1306UUAGGAGUGAGGUACCUGG 1620 143858 AD-71932 A-143861AACCUGUGACAAUGACAGAdTdT  993 A- UCUGUCAUUGUCACAGGUUdTdT 1307AACCUGUGACAAUGACAGC 1621 143862 AD-71933 A-143863GCAGACAAAUUGGUUUUCUdTdT  994 A- AGAAAACCAAUUUGUCUGCdTdT 1308GCAGACAAAUUGGUUUUCU 1622 143864 AD-71934 A-143865UUUGUGAAUGGCAGAAAGAdTdT  995 A- UCUUUCUGCCAUUCACAAAdTdT 1309UUUGUGAAUGGCAGAAAGG 1623 143866 AD-71935 A-143867AAGGUGGUGGAGAAAAAUAdTdT  996 A- UAUUUUUCUCCACCACCUUdTdT 1310AAGGUGGUGGAGAAAAAUG 1624 143868 AD-71936 A-143871CCUUUUGGCCUACCUGAGAdTdT  997 A- UCUCAGGUAGGCCAAAAGGdTdT 1311CCUUUUGGCCUACCUGAGA 1625 143872 AD-71937 A-143873AAGAAAGUUGGGGCUGAGUdTdT  998 A- ACUCAGCCCCAACUUUCUUdTdT 1312AAGAAAGUUGGGGCUGAGU 1626 143874 AD-71938 A-143875AGUGGAACCAAGCUCGGCUdTdT  999 A- AGCCGAGCUUGGUUCCACUdTdT 1313AGUGGAACCAAGCUCGGCU 1627 143876 AD-71939 A-143877CUGUGGAGAGGGGGGCUGAdTdT 1000 A- UCAGCCCCCCUCUCCACAGdTdT 1314CUGUGGAGAGGGGGGCUGC 1628 143878 AD-71940 A-143879CGGGGCUUGCACAGUGAUAdTdT 1001 A- UAUCACUGUGCAAGCCCCGdTdT 1315CGGGGCUUGCACAGUGAUG 1629 143880 AD-71941 A-143881AUGCUCUCCAAGUAUGAUAdTdT 1002 A- UAUCAUACUUGGAGAGCAUdTdT 1316AUGCUCUCCAAGUAUGAUC 1630 143882 AD-71942 A-143885UCGUCCACUUUUCUGCCAAdTdT 1003 A- UUGGCAGAAAAGUGGACGAdTdT 1317UCGUCCACUUUUCUGCCAA 1631 143886 AD-71943 A-143887AUGCCUGCCUGGCCCCCAUdTdT 1004 A- AUGGGGGCCAGGCAGGCAUdTdT 1318AUGCCUGCCUGGCCCCCAU 1632 143888 AD-71944 A-143889AUCUGCUCCUUGCACCAUAdTdT 1005 A- UAUGGUGCAAGGAGCAGAUdTdT 1319AUCUGCUCCUUGCACCAUG 1633 143890 AD-71945 A-143891UGUUGCAGUGACAACUGUAdTdT 1006 A- UACAGUUGUCACUGCAACAdTdT 1320UGUUGCAGUGACAACUGUG 1634 143892 AD-71946 A-143893UGGAAGGAAUAGGAAGCAAdTdT 1007 A- UUGCUUCCUAUUCCUUCCAdTdT 1321UGGAAGGAAUAGGAAGCAC 1635 143894 AD-71947 A-143895CACCAAGACGAGGCUGCAUdTdT 1008 A- AUGCAGCCUCGUCUUGGUGdTdT 1322CACCAAGACGAGGCUGCAU 1636 143896 AD-71948 A-143897UCCUGUGCAGGAGAGAAUUdTdT 1009 A- AAUUCUCUCCUGCACAGGAdTdT 1323UCCUGUGCAGGAGAGAAUU 1637 143898 AD-71949 A-143899AUUGCCAAAAGCCACGGCUdTdT 1010 A- AGCCGUGGCUUUUGGCAAUdTdT 1324AUUGCCAAAAGCCACGGCU 1638 143900 AD-71950 A-143901UCCCAGUGCGGGUUCUGCAdTdT 1011 A- UGCAGAACCCGCACUGGGAdTdT 1325UCCCAGUGCGGGUUCUGCA 1639 143902 AD-71951 A-143903UGCACCCCUGGCAUCGUCAdTdT 1012 A- UGACGAUGCCAGGGGUGCAdTdT 1326UGCACCCCUGGCAUCGUCA 1640 143904 AD-71952 A-143905UGAGUAUGUACACACUGCUdTdT 1013 A- AGCAGUGUGUACAUACUCAdTdT 1327UGAGUAUGUACACACUGCU 1641 143906 AD-71953 A-143907UGCUCCGGAAUCAGCCCGAdTdT 1014 A- UCGGGCUGAUUCCGGAGCAdTdT 1328UGCUCCGGAAUCAGCCCGA 1642 143908 AD-71954 A-143909AGCCCACCAUGGAGGAGAUdTdT 1015 A- AUCUCCUCCAUGGUGGGCUdTdT 1329AGCCCACCAUGGAGGAGAU 1643 143910 AD-71955 A-143911UUGAGAAUGCCUUCCAAGAdTdT 1016 A- UCUUGGAAGGCAUUCUCAAdTdT 1330UUGAGAAUGCCUUCCAAGG 1644 143912 AD-71956 A-143913AAGGAAAUCUGUGCCGCUAdTdT 1017 A- UAGCGGCACAGAUUUCCUUdTdT 1331AAGGAAAUCUGUGCCGCUG 1645 143914 AD-71957 A-143915CACAGGCUACAGACCCAUAdTdT 1018 A- UAUGGGUCUGUAGCCUGUGdTdT 1332CACAGGCUACAGACCCAUC 1646 143916 AD-71958 A-143917CAUCCUCCAGGGCUUCCGAdTdT 1019 A- UCGGAAGCCCUGGAGGAUGdTdT 1333CAUCCUCCAGGGCUUCCGG 1647 143918 AD-71959 A-143919ACCUUUGCCAGGGAUGGUAdTdT 1020 A- UACCAUCCCUGGCAAAGGUdTdT 1334ACCUUUGCCAGGGAUGGUG 1648 143920 AD-71960 A-143921UGGAUGCUGUGGAGGAGAUdTdT 1021 A- AUCUCCUCCACAGCAUCCAdTdT 1335UGGAUGCUGUGGAGGAGAU 1649 143922 AD-71961 A-143923GAUGGGAAUAAUCCAAAUUdTdT 1022 A- AAUUUGGAUUAUUCCCAUCdTdT 1336GAUGGGAAUAAUCCAAAUU 1650 143924 AD-71962 A-143927AGAAAGACCACUCAGUCAAdTdT 1023 A- UUGACUGAGUGGUCUUUCUdTdT 1337AGAAAGACCACUCAGUCAG 1651 143928 AD-71963 A-143929AGCCUCUCGCCAUCUUUAUdTdT 1024 A- AUAAAGAUGGCGAGAGGCUdTdT 1338AGCCUCUCGCCAUCUUUAU 1652 143930 AD-71964 A-143931UAUUCAAACCAGAGGAGUUdTdT 1025 A- AACUCCUCUGGUUUGAAUAdTdT 1339UAUUCAAACCAGAGGAGUU 1653 143932 AD-71965 A-143933UUCACGCCCCUGGAUCCAAdTdT 1026 A- UUGGAUCCAGGGGCGUGAAdTdT 1340UUCACGCCCCUGGAUCCAA 1654 143934 AD-71966 A-143935AACCCAGGAGCCCAUUUUUdTdT 1027 A- AAAAAUGGGCUCCUGGGUUdTdT 1341AACCCAGGAGCCCAUUUUU 1655 143936 AD-71967 A-143937UUCCCCCAGAGUUGCUGAAdTdT 1028 A- UUCAGCAACUCUGGGGGAAdTdT 1342UUCCCCCAGAGUUGCUGAG 1656 143938 AD-71968 A-143939AGGCUGAAAGACACUCCUAdTdT 1029 A- UAGGAGUGUCUUUCAGCCUdTdT 1343AGGCUGAAAGACACUCCUC 1657 143940 AD-71969 A-143941UCGGAAGCAGCUGCGAUUUdTdT 1030 A- AAAUCGCAGCUGCUUCCGAdTdT 1344UCGGAAGCAGCUGCGAUUU 1658 143942 AD-71970 A-143943UUGAAGGGGAGCGUGUGAAdTdT 1031 A- UUCACACGCUCCCCUUCAAdTdT 1345UUGAAGGGGAGCGUGUGAC 1659 143944 AD-71971 A-143945CGUGGAUACAGGCCUCAAAdTdT 1032 A- UUUGAGGCCUGUAUCCACGdTdT 1346CGUGGAUACAGGCCUCAAC 1660 143946 AD-71972 A-143947AACCCUCAAGGAGCUGCUAdTdT 1033 A- UAGCAGCUCCUUGAGGGUUdTdT 1347AACCCUCAAGGAGCUGCUG 1661 143948 AD-71973 A-143949UGGACCUCAAGGCUCAGCAdTdT 1034 A- UGCUGAGCCUUGAGGUCCAdTdT 1348UGGACCUCAAGGCUCAGCA 1662 143950 AD-71974 A-143951ACCCUGACGCCAAGCUGGUdTdT 1035 A- ACCAGCUUGGCGUCAGGGUdTdT 1349ACCCUGACGCCAAGCUGGU 1663 143952 AD-71975 A-143953UCGUGGGGAACACGGAGAUdTdT 1036 A- AUCUCCGUGUUCCCCACGAdTdT 1350UCGUGGGGAACACGGAGAU 1664 143954 AD-71976 A-143955AUUGGCAUUGAGAUGAAGUdTdT 1037 A- ACUUCAUCUCAAUGCCAAUdTdT 1351AUUGGCAUUGAGAUGAAGU 1665 143956 AD-71977 A-143957AAGUUCAAGAAUAUGCUGUdTdT 1038 A- ACAGCAUAUUCUUGAACUUdTdT 1352AAGUUCAAGAAUAUGCUGU 1666 143958 AD-71978 A-143959UUUCCUAUGAUUGUCUGCAdTdT 1039 A- UGCAGACAAUCAUAGGAAAdTdT 1353UUUCCUAUGAUUGUCUGCC 1667 143960 AD-71979 A-143961CCCAGCCUGGAUCCCUGAAdTdT 1040 A- UUCAGGGAUCCAGGCUGGGdTdT 1354CCCAGCCUGGAUCCCUGAG 1668 143962 AD-71980 A-143963AGCUGAAUUCGGUAGAACAdTdT 1041 A- UGUUCUACCGAAUUCAGCUdTdT 1355AGCUGAAUUCGGUAGAACA 1669 143964 AD-71981 A-143965CAUGGACCCGACGGUAUCUdTdT 1042 A- AGAUACCGUCGGGUCCAUGdTdT 1356CAUGGACCCGACGGUAUCU 1670 143966 AD-71982 A-143967UCUCCUUUGGAGCUGCUUAdTdT 1043 A- UAAGCAGCUCCAAAGGAGAdTdT 1357UCUCCUUUGGAGCUGCUUG 1671 143968 AD-71983 A-143969UGCCCCCUGAGCAUUGUGAdTdT 1044 A- UCACAAUGCUCAGGGGGCAdTdT 1358UGCCCCCUGAGCAUUGUGG 1672 143970 AD-71984 A-143971AAAAAACCCUGGUGGAUGAdTdT 1045 A- UCAUCCACCAGGGUUUUUUdTdT 1359AAAAAACCCUGGUGGAUGC 1673 143972 AD-71985 A-143973UGCUGUUGCUAAGCUUCCUdTdT 1046 A- AGGAAGCUUAGCAACAGCAdTdT 1360UGCUGUUGCUAAGCUUCCU 1674 143974 AD-71986 A-143975CCUGCCCAAAAGACAGAGAdTdT 1047 A- UCUCUGUCUUUUGGGCAGGdTdT 1361CCUGCCCAAAAGACAGAGG 1675 143976 AD-71987 A-143977UGUUCAGAGGGGUCCUGGAdTdT 1048 A- UCCAGGACCCCUCUGAACAdTdT 1362UGUUCAGAGGGGUCCUGGA 1676 143978 AD-71988 A-143979UGGAGCAGCUGCGCUGGUUdTdT 1049 A- AACCAGCGCAGCUGCUCCAdTdT 1363UGGAGCAGCUGCGCUGGUU 1677 143980 AD-71989 A-143981UUUGCUGGGAAGCAAGUCAdTdT 1050 A- UGACUUGCUUCCCAGCAAAdTdT 1364UUUGCUGGGAAGCAAGUCA 1678 143982 AD-71990 A-143983CAAGUCUGUGGCGUCCGUUdTdT 1051 A- AACGGACGCCACAGACUUGdTdT 1365CAAGUCUGUGGCGUCCGUU 1679 143984 AD-71991 A-143985UGGAGGGAACAUCAUCACUdTdT 1052 A- AGUGAUGAUGUUCCCUCCAdTdT 1366UGGAGGGAACAUCAUCACU 1680 143986 AD-71992 A-143987UGCCAGCCCCAUCUCCGAAdTdT 1053 A- UUCGGAGAUGGGGCUGGCAdTdT 1367UGCCAGCCCCAUCUCCGAC 1681 143988 AD-71993 A-143989ACCUCAACCCCGUGUUCAUdTdT 1054 A- AUGAACACGGGGUUGAGGUdTdT 1368ACCUCAACCCCGUGUUCAU 1682 143990 AD-71994 A-143991UCAUGGCCAGUGGGGCCAAdTdT 1055 A- UUGGCCCCACUGGCCAUGAdTdT 1369UCAUGGCCAGUGGGGCCAA 1683 143992 AD-71995 A-143993AAGCUGACACUUGUGUCCAdTdT 1056 A- UGGACACAAGUGUCAGCUUdTdT 1370AAGCUGACACUUGUGUCCA 1684 143994 AD-71996 A-143995CAGAGGCACCAGGAGAACUdTdT 1057 A- AGUUCUCCUGGUGCCUCUGdTdT 1371CAGAGGCACCAGGAGAACU 1685 143996 AD-71997 A-143997UGUCCAGAUGGACCACACAdTdT 1058 A- UGUGUGGUCCAUCUGGACAdTdT 1372UGUCCAGAUGGACCACACC 1686 143998 AD-71998 A-143999CCUUCUUCCCUGGCUACAAdTdT 1059 A- UUGUAGCCAGGGAAGAAGGdTdT 1373CCUUCUUCCCUGGCUACAG 1687 144000 AD-71999 A-144001AGAAAGACCCUGCUGAGCAdTdT 1060 A- UGCUCAGCAGGGUCUUUCUdTdT 1374AGAAAGACCCUGCUGAGCC 1688 144002 AD-72000 A-144003CCGGAGGAGAUACUGCUCUdTdT 1061 A- AGAGCAGUAUCUCCUCCGGdTdT 1375CCGGAGGAGAUACUGCUCU 1689 144004 AD-72001 A-144005CUCUCCAUAGAGAUCCCCUdTdT 1062 A- AGGGGAUCUCUAUGGAGAGdTdT 1376CUCUCCAUAGAGAUCCCCU 1690 144006 AD-72002 A-144007UACAGCAGGGAGGGGGAGUdTdT 1063 A- ACUCCCCCUCCCUGCUGUAdTdT 1377UACAGCAGGGAGGGGGAGU 1691 144008 AD-72003 A-144009AGUAUUUCUCAGCAUUCAAdTdT 1064 A- UUGAAUGCUGAGAAAUACUdTdT 1378AGUAUUUCUCAGCAUUCAA 1692 144010 AD-72004 A-144011AAGCAGGCCUCCCGGAGAAdTdT 1065 A- UUCUCCGGGAGGCCUGCUUdTdT 1379AAGCAGGCCUCCCGGAGAG 1693 144012 AD-72005 A-144013AAGAUGACAUUGCCAAGGUdTdT 1066 A- ACCUUGGCAAUGUCAUCUUdTdT 1380AAGAUGACAUUGCCAAGGU 1694 144014 AD-72006 A-144015GGUAACCAGUGGCAUGAGAdTdT 1067 A- UCUCAUGCCACUGGUUACCdTdT 1381GGUAACCAGUGGCAUGAGA 1695 144016 AD-72007 A-144017AGAGUUUUAUUCAAGCCAAdTdT 1068 A- UUGGCUUGAAUAAAACUCUdTdT 1382AGAGUUUUAUUCAAGCCAG 1696 144018 AD-72008 A-144019AGGAACCACAGAGGUACAAdTdT 1069 A- UUGUACCUCUGUGGUUCCUdTdT 1383AGGAACCACAGAGGUACAG 1697 144020 AD-72009 A-144021AGGAGCUGGCCCUUUGCUAdTdT 1070 A- UAGCAAAGGGCCAGCUCCUdTdT 1384AGGAGCUGGCCCUUUGCUA 1698 144022 AD-72010 A-144023UAUGGUGGAAUGGCCAACAdTdT 1071 A- UGUUGGCCAUUCCACCAUAdTdT 1385UAUGGUGGAAUGGCCAACA 1699 144024 AD-72011 A-144025AGAACCAUCUCAGCCCUCAdTdT 1072 A- UGAGGGCUGAGAUGGUUCUdTdT 1386AGAACCAUCUCAGCCCUCA 1700 144026 AD-72012 A-144027AAGACCACUCAGAGGCAGAdTdT 1073 A- UCUGCCUCUGAGUGGUCUUdTdT 1387AAGACCACUCAGAGGCAGC 1701 144028 AD-72013 A-144029CAGCUUUCCAAGCUCUGGAdTdT 1074 A- UCCAGAGCUUGGAAAGCUGdTdT 1388CAGCUUUCCAAGCUCUGGA 1702 144030 AD-72014 A-144031AGGAGGAGCUGCUGCAGGAdTdT 1075 A- UCCUGCAGCAGCUCCUCCUdTdT 1389AGGAGGAGCUGCUGCAGGA 1703 144032 AD-72015 A-144033AGGACGUGUGUGCAGGACUdTdT 1076 A- AGUCCUGCACACACGUCCUdTdT 1390AGGACGUGUGUGCAGGACU 1704 144034 AD-72016 A-144035UGGCAGAGGAGCUGCAUCUdTdT 1077 A- AGAUGCAGCUCCUCUGCCAdTdT 1391UGGCAGAGGAGCUGCAUCU 1705 144036 AD-72017 A-144037UCUGCCUCCCGAUGCCCCUdTdT 1078 A- AGGGGCAUCGGGAGGCAGAdTdT 1392UCUGCCUCCCGAUGCCCCU 1706 144038 AD-72018 A-144039GGUGGCAUGGUGGACUUCAdTdT 1079 A- UGAAGUCCACCAUGCCACCdTdT 1393GGUGGCAUGGUGGACUUCC 1707 144040 AD-72019 A-144041UUCCGGUGCACCCUCACCAdTdT 1080 A- UGGUGAGGGUGCACCGGAAdTdT 1394UUCCGGUGCACCCUCACCC 1708 144042 AD-71752 A-144043UCAGCUUCUUCUUCAAGUUdTdT 1081 A- AACUUGAAGAAGAAGCUGAdTdT 1395UCAGCUUCUUCUUCAAGUU 1709 144044 AD-71753 A-144045UUCUACCUGACAGUCCUUAdTdT 1082 A- UAAGGACUGUCAGGUAGAAdTdT 1396UUCUACCUGACAGUCCUUC 1710 144046 AD-71754 A-144049GAGAACCUGGAAGACAAGUdTdT 1083 A- ACUUGUCUUCCAGGUUCUCdTdT 1397GAGAACCUGGAAGACAAGU 1711 144050 AD-71755 A-144051UGUGGUAAACUGGACCCCAdTdT 1084 A- UGGGGUCCAGUUUACCACAdTdT 1398UGUGGUAAACUGGACCCCA 1712 144052 AD-71756 A-144053CACUUUCGCCAGUGCAACUdTdT 1085 A- AGUUGCACUGGCGAAAGUGdTdT 1399CACUUUCGCCAGUGCAACU 1713 144054 AD-71757 A-144055ACUUUACUGUUUCAGAAAGdTdT 1086 A- CUUUCUGAAACAGUAAAGUdTdT 1400ACUUUACUGUUUCAGAAAG 1714 144056 AD-71758 A-144057AAGACCCCCCAGCCGAUGUdTdT 1087 A- ACAUCGGCUGGGGGGUCUUdTdT 1401AAGACCCCCCAGCCGAUGU 1715 144058 AD-71759 A-144059UCCAGCUCUUCCAAGAGGUdTdT 1088 A- ACCUCUUGGAAGAGCUGGAdTdT 1402UCCAGCUCUUCCAAGAGGU 1716 144060 AD-71760 A-144061UGCCCAAGGGUCAGUCUGAdTdT 1089 A- UCAGACUGACCCUUGGGCAdTdT 1403UGCCCAAGGGUCAGUCUGA 1717 144062 AD-71761 A-144063GAGGAGGACAUGGUGGGCAdTdT 1090 A- UGCCCACCAUGUCCUCCUCdTdT 1404GAGGAGGACAUGGUGGGCC 1718 144064 AD-71762 A-144065GCCGGCCCCUGCCCCACCUdTdT 1091 A- AGGUGGGGCAGGGGCCGGCdTdT 1405GCCGGCCCCUGCCCCACCU 1719 144066 AD-71763 A-144067CCUGGCAGCGGACAUGCAAdTdT 1092 A- UUGCAUGUCCGCUGCCAGGdTdT 1406CCUGGCAGCGGACAUGCAG 1720 144068 AD-71764 A-144069AGGCCUCUGGUGAGGCCGUdTdT 1093 A- ACGGCCUCACCAGAGGCCUdTdT 1407AGGCCUCUGGUGAGGCCGU 1721 144070 AD-71765 A-144071UGUACUGUGACGACAUUCAdTdT 1094 A- UGAAUGUCGUCACAGUACAdTdT 1408UGUACUGUGACGACAUUCC 1722 144072 AD-71766 A-144073CUCGCUACGAGAAUGAGCUdTdT 1095 A- AGCUCAUUCUCGUAGCGAGdTdT 1409CUCGCUACGAGAAUGAGCU 1723 144074 AD-71767 A-144075CUGUCUCUCCGGCUGGUCAdTdT 1096 A- UGACCAGCCGGAGAGACAGdTdT 1410CUGUCUCUCCGGCUGGUCA 1724 144076 AD-71768 A-144079CCACGCCAAGAUCAAGUCAdTdT 1097 A- UGACUUGAUCUUGGCGUGGdTdT 1411CCACGCCAAGAUCAAGUCC 1725 144080 AD-71769 A-144081CAUAGAUACAUCAGAAGCUdTdT 1098 A- AGCUUCUGAUGUAUCUAUGdTdT 1412CAUAGAUACAUCAGAAGCU 1726 144082 AD-71770 A-144085UUUGUUUGUUUCAUUUCCAdTdT 1099 A- UGGAAAUGAAACAAACAAAdTdT 1413UUUGUUUGUUUCAUUUCCG 1727 144086 AD-71771 A-144087GCUGAUGAUGUUCCUGGGAdTdT 1100 A- UCCCAGGAACAUCAUCAGCdTdT 1414GCUGAUGAUGUUCCUGGGA 1728 144088 AD-71772 A-144093CAGUCUUUGCGAAGGAUAAdTdT 1101 A- UUAUCCUUCGCAAAGACUGdTdT 1415CAGUCUUUGCGAAGGAUAA 1729 144094 AD-71773 A-144095AAGGUUACUUGUGUUGGGAdTdT 1102 A- UCCCAACACAAGUAACCUUdTdT 1416AAGGUUACUUGUGUUGGGC 1730 144096 AD-71774 A-144097CAUAUCAUUGGUGCUGUGAdTdT 1103 A- UCACAGCACCAAUGAUAUGdTdT 1417CAUAUCAUUGGUGCUGUGG 1731 144098 AD-71775 A-144099UGGUUGCUGACACCCCGGAdTdT 1104 A- UCCGGGGUGUCAGCAACCAdTdT 1418UGGUUGCUGACACCCCGGA 1732 144100 AD-71776 A-144101ACACACACAGAGAGCUGCAdTdT 1105 A- UGCAGCUCUCUGUGUGUGUdTdT 1419ACACACACAGAGAGCUGCC 1733 144102 AD-71777 A-144103GCCCAAGGGGUGAAAAUCAdTdT 1106 A- UGAUUUUCACCCCUUGGGCdTdT 1420GCCCAAGGGGUGAAAAUCA 1734 144104 AD-71778 A-144105UCACCUAUGAAGAACUACAdTdT 1107 A- UGUAGUUCUUCAUAGGUGAdTdT 1421UCACCUAUGAAGAACUACC 1735 144106 AD-71779 A-144109GAGGAUGCUAUAAAGAACAdTdT 1108 A- UGUUCUUUAUAGCAUCCUCdTdT 1422GAGGAUGCUAUAAAGAACA 1736 144110 AD-71780 A-144111CAACUCCUUUUAUGGACCUdTdT 1109 A- AGGUCCAUAAAAGGAGUUGdTdT 1423CAACUCCUUUUAUGGACCU 1737 144112 AD-71781 A-144113CCUGAGCUGAAGAUCGAGAdTdT 1110 A- UCUCGAUCUUCAGCUCAGGdTdT 1424CCUGAGCUGAAGAUCGAGA 1738 144114 AD-71782 A-144115AAAGGGGACCUAAAGAAGAdTdT 1111 A- UCUUCUUUAGGUCCCCUUUdTdT 1425AAAGGGGACCUAAAGAAGG 1739 144116 AD-71783 A-144117AGGGGUUUUCCGAAGCAGAdTdT 1112 A- UCUGCUUCGGAAAACCCCUdTdT 1426AGGGGUUUUCCGAAGCAGA 1740 144118 AD-71784 A-144119UAAUGUUGUGUCAGGGGAAdTdT 1113 A- UUCCCCUGACACAACAUUAdTdT 1427UAAUGUUGUGUCAGGGGAG 1741 144120 AD-71785 A-144121AGAUAUACAUCGGUGGCCAdTdT 1114 A- UGGCCACCGAUGUAUAUCUdTdT 1428AGAUAUACAUCGGUGGCCA 1742 144122 AD-71786 A-144123GCCAAGAGCACUUCUACCUdTdT 1115 A- AGGUAGAAGUGCUCUUGGCdTdT 1429GCCAAGAGCACUUCUACCU 1743 144124 AD-71787 A-144125GGAGACUCACUGCACCAUUdTdT 1116 A- AAUGGUGCAGUGAGUCUCCdTdT 1430GGAGACUCACUGCACCAUU 1744 144126 AD-71788 A-144127UUGCUGUUCCAAAAGGCGAdTdT 1117 A- UCGCCUUUUGGAACAGCAAdTdT 1431UUGCUGUUCCAAAAGGCGA 1745 144128 AD-71789 A-144129CGAGGCAGGGGAGAUGGAAdTdT 1118 A- UUCCAUCUCCCCUGCCUCGdTdT 1432CGAGGCAGGGGAGAUGGAG 1746 144130 AD-71790 A-144131AGCUCUUUGUGUCUACACAdTdT 1119 A- UGUGUAGACACAAAGAGCUdTdT 1433AGCUCUUUGUGUCUACACA 1747 144132 AD-71791 A-144133CAGAACACCAUGAAGACCAdTdT 1120 A- UGGUCUUCAUGGUGUUCUGdTdT 1434CAGAACACCAUGAAGACCC 1748 144134 AD-71792 A-144135CAGAGCUUUGUUGCAAAAAdTdT 1121 A- UUUUUGCAACAAAGCUCUGdTdT 1435CAGAGCUUUGUUGCAAAAA 1749 144136 AD-71793 A-144137AAAAUGUUGGGGGUUCCAAdTdT 1122 A- UUGGAACCCCCAACAUUUUdTdT 1436AAAAUGUUGGGGGUUCCAG 1750 144138 AD-71794 A-144139AGCAAACCGGAUUGUGGUUdTdT 1123 A- AACCACAAUCCGGUUUGCUdTdT 1437AGCAAACCGGAUUGUGGUU 1751 144140 AD-71795 A-144141UUCGAGUGAAGAGAAUGGAdTdT 1124 A- UCCAUUCUCUUCACUCGAAdTdT 1438UUCGAGUGAAGAGAAUGGG 1752 144142 AD-71796 A-144143AGGAGGCUUUGGAGGCAAAdTdT 1125 A- UUUGCCUCCAAAGCCUCCUdTdT 1439AGGAGGCUUUGGAGGCAAG 1753 144144 AD-71797 A-144145AAGGAGACCCGGAGCACUAdTdT 1126 A- UAGUGCUCCGGGUCUCCUUdTdT 1440AAGGAGACCCGGAGCACUG 1754 144146 AD-71798 A-144147UGUGGUGUCCACGGCAGUAdTdT 1127 A- UACUGCCGUGGACACCACAdTdT 1441UGUGGUGUCCACGGCAGUG 1755 144148 AD-71799 A-144149UGGCCCUGGCUGCAUAUAAdTdT 1128 A- UUAUAUGCAGCCAGGGCCAdTdT 1442UGGCCCUGGCUGCAUAUAA 1756 144150 AD-71800 A-144153UGCGAUGCAUGCUGGACCAdTdT 1129 A- UGGUCCAGCAUGCAUCGCAdTdT 1443UGCGAUGCAUGCUGGACCG 1757 144154 AD-71801 A-144155CGUGAUGAGGACAUGCUGAdTdT 1130 A- UCAGCAUGUCCUCAUCACGdTdT 1444CGUGAUGAGGACAUGCUGA 1758 144156 AD-71802 A-144157UAACUGGUGGCAGACAUCAdTdT 1131 A- UGAUGUCUGCCACCAGUUAdTdT 1445UAACUGGUGGCAGACAUCC 1759 144158 AD-71803 A-144159UCCCUUCCUGGCCAGAUAAdTdT 1132 A- UUAUCUGGCCAGGAAGGGAdTdT 1446UCCCUUCCUGGCCAGAUAC 1760 144160 AD-71804 A-144161UACAAGGUUGGCUUCAUGAdTdT 1133 A- UCAUGAAGCCAACCUUGUAdTdT 1447UACAAGGUUGGCUUCAUGA 1761 144162 AD-71805 A-144163AGACUGGGACAGUUGUGGAdTdT 1134 A- UCCACAACUGUCCCAGUCUdTdT 1448AGACUGGGACAGUUGUGGC 1762 144164 AD-71806 A-144165GCUCUUGAGGUGGACCACUdTdT 1135 A- AGUGGUCCACCUCAAGAGCdTdT 1449GCUCUUGAGGUGGACCACU 1763 144166 AD-71807 A-144167ACUUCAGCAAUGUGGGGAAdTdT 1136 A- UUCCCCACAUUGCUGAAGUdTdT 1450ACUUCAGCAAUGUGGGGAA 1764 144168 AD-71808 A-144169GAACACCCAGGAUCUCUCUdTdT 1137 A- AGAGAGAUCCUGGGUGUUCdTdT 1451GAACACCCAGGAUCUCUCU 1765 144170 AD-71809 A-144171CAGAGUAUUAUGGAACGAAdTdT 1138 A- UUCGUUCCAUAAUACUCUGdTdT 1452CAGAGUAUUAUGGAACGAG 1766 144172 AD-71810 A-144173AGCUUUAUUCCACAUGGAAdTdT 1139 A- UUCCAUGUGGAAUAAAGCUdTdT 1453AGCUUUAUUCCACAUGGAC 1767 144174 AD-71811 A-144175ACAACUGCUAUAAAAUCCAdTdT 1140 A- UGGAUUUUAUAGCAGUUGUdTdT 1454ACAACUGCUAUAAAAUCCC 1768 144176 AD-71812 A-144177UCCCCAACAUCCGGGGCAAdTdT 1141 A- UUGCCCCGGAUGUUGGGGAdTdT 1455UCCCCAACAUCCGGGGCAC 1769 144178 AD-71813 A-144179UGGGCGGCUGUGCAAAACAdTdT 1142 A- UGUUUUGCACAGCCGCCCAdTdT 1456UGGGCGGCUGUGCAAAACC 1770 144180 AD-71814 A-144181AACCAACCUUCCCUCCAAAdTdT 1143 A- UUUGGAGGGAAGGUUGGUUdTdT 1457AACCAACCUUCCCUCCAAC 1771 144182 AD-71815 A-144183ACACGGCCUUCCGGGGCUUdTdT 1144 A- AAGCCCCGGAAGGCCGUGUdTdT 1458ACACGGCCUUCCGGGGCUU 1772 144184 AD-71816 A-144185UUGGGGGGCCCCAGGGGAUdTdT 1145 A- AUCCCCUGGGGCCCCCCAAdTdT 1459UUGGGGGGCCCCAGGGGAU 1773 144186 AD-71817 A-144187AUGCUCAUUGCCGAGUGCUdTdT 1146 A- AGCACUCGGCAAUGAGCAUdTdT 1460AUGCUCAUUGCCGAGUGCU 1774 144188 AD-71818 A-144189UGGAUGAGUGAAGUUGCAAdTdT 1147 A- UUGCAACUUCACUCAUCCAdTdT 1461UGGAUGAGUGAAGUUGCAG 1775 144190 AD-71819 A-144191AGUGACCUGUGGGAUGCCUdTdT 1148 A- AGGCAUCCCACAGGUCACUdTdT 1462AGUGACCUGUGGGAUGCCU 1776 144192 AD-71820 A-144193CCUGCAGAGGAGGUGCGGAdTdT 1149 A- UCCGCACCUCCUCUGCAGGdTdT 1463CCUGCAGAGGAGGUGCGGA 1777 144194 AD-71821 A-144195AGAAAAAACCUGUACAAAGdTdT 1150 A- CUUUGUACAGGUUUUUUCUdTdT 1464AGAAAAAACCUGUACAAAG 1778 144196 AD-71822 A-144197AAAGAAGGGGACCUGACAAdTdT 1151 A- UUGUCAGGUCCCCUUCUUUdTdT 1465AAAGAAGGGGACCUGACAC 1779 144198 AD-71823 A-144199ACACUUCAACCAGAAGCUUdTdT 1152 A- AAGCUUCUGGUUGAAGUGUdTdT 1466ACACUUCAACCAGAAGCUU 1780 144200 AD-71824 A-144201UUGAGGGUUUCACCUUGCAdTdT 1153 A- UGCAAGGUGAAACCCUCAAdTdT 1467UUGAGGGUUUCACCUUGCC 1781 144202 AD-71825 A-144203CAGAUGCUGGGAAGAAUGAdTdT 1154 A- UCAUUCUUCCCAGCAUCUGdTdT 1468CAGAUGCUGGGAAGAAUGC 1782 144204 AD-71826 A-144205UGCCUAGCAAGCUCUCAGUdTdT 1155 A- ACUGAGAGCUUGCUAGGCAdTdT 1469UGCCUAGCAAGCUCUCAGU 1783 144206 AD-71827 A-144207UAUCAUGCUCGGAAGAGUAdTdT 1156 A- UACUCUUCCGAGCAUGAUAdTdT 1470UAUCAUGCUCGGAAGAGUG 1784 144208 AD-71828 A-144209AGUGAGGUUGACAAGUUCAdTdT 1157 A- UGAACUUGUCAACCUCACUdTdT 1471AGUGAGGUUGACAAGUUCA 1785 144210 AD-71829 A-144211AACAAGGAGAAUUGUUGGAdTdT 1158 A- UCCAACAAUUCUCCUUGUUdTdT 1472AACAAGGAGAAUUGUUGGA 1786 144212 AD-71830 A-144213AAAAAGAGAGGAUUGUGCAdTdT 1159 A- UGCACAAUCCUCUCUUUUUdTdT 1473AAAAAGAGAGGAUUGUGCA 1787 144214 AD-71831 A-144215CAUAAUUCCCACCAAGUUUdTdT 1160 A- AAACUUGGUGGGAAUUAUGdTdT 1474CAUAAUUCCCACCAAGUUU 1788 144216 AD-71832 A-144217UUUGGAAUAAGCUUUACAAdTdT 1161 A- UUGUAAAGCUUAUUCCAAAdTdT 1475UUUGGAAUAAGCUUUACAG 1789 144218 AD-71833 A-144219AGUUCCUUUUCUGAAUCAAdTdT 1162 A- UUGAUUCAGAAAAGGAACUdTdT 1476AGUUCCUUUUCUGAAUCAG 1790 144220 AD-71834 A-144221AGGCAGGAGCCCUACUUCAdTdT 1163 A- UGAAGUAGGGCUCCUGCCUdTdT 1477AGGCAGGAGCCCUACUUCA 1791 144222 AD-71835 A-144223CAUGUGUACACAGAUGGCUdTdT 1164 A- AGCCAUCUGUGUACACAUGdTdT 1478CAUGUGUACACAGAUGGCU 1792 144224 AD-71836 A-144225UCUGUGCUGCUGACCCACAdTdT 1165 A- UGUGGGUCAGCAGCACAGAdTdT 1479UCUGUGCUGCUGACCCACG 1793 144226 AD-71837 A-144229GGCCAAGGCCUUCAUACCAdTdT 1166 A- UGGUAUGAAGGCCUUGGCCdTdT 1480GGCCAAGGCCUUCAUACCA 1794 144230 AD-71838 A-144231AAAAUGGUCCAGGUGGCCAdTdT 1167 A- UGGCCACCUGGACCAUUUUdTdT 1481AAAAUGGUCCAGGUGGCCA 1795 144232 AD-71839 A-144233GCCAGUAGAGCUCUGAAAAdTdT 1168 A- UUUUCAGAGCUCUACUGGCdTdT 1482GCCAGUAGAGCUCUGAAAA 1796 144234 AD-71840 A-144235AAUCCCCACCUCUAAGAUUdTdT 1169 A- AAUCUUAGAGGUGGGGAUUdTdT 1483AAUCCCCACCUCUAAGAUU 1797 144236 AD-71841 A-144237UAUAUCAGCGAGACAAGCAdTdT 1170 A- UGCUUGUCUCGCUGAUAUAdTdT 1484UAUAUCAGCGAGACAAGCA 1798 144238 AD-71842 A-144239AGCACUAACACUGUGCCCAdTdT 1171 A- UGGGCACAGUGUUAGUGCUdTdT 1485AGCACUAACACUGUGCCCA 1799 144240 AD-71843 A-144241CAACACCUCUCCCACGGCUdTdT 1172 A- AGCCGUGGGAGAGGUGUUGdTdT 1486CAACACCUCUCCCACGGCU 1800 144242 AD-71844 A-144243UGCCUCUGUCAGCGCUGAAdTdT 1173 A- UUCAGCGCUGACAGAGGCAdTdT 1487UGCCUCUGUCAGCGCUGAC 1801 144244 AD-71845 A-144245ACCUCAAUGGACAGGCCGUdTdT 1174 A- ACGGCCUGUCCAUUGAGGUdTdT 1488ACCUCAAUGGACAGGCCGU 1802 144246 AD-71846 A-144247GUCUAUGCGGCUUGUCAGAdTdT 1175 A- UCUGACAAGCCGCAUAGACdTdT 1489GUCUAUGCGGCUUGUCAGA 1803 144248 AD-71847 A-144249AGACCAUCUUGAAAAGGCUdTdT 1176 A- AGCCUUUUCAAGAUGGUCUdTdT 1490AGACCAUCUUGAAAAGGCU 1804 144250 AD-71848 A-144251UGGAACCCUACAAGAAGAAdTdT 1177 A- UUCUUCUUGUAGGGUUCCAdTdT 1491UGGAACCCUACAAGAAGAA 1805 144252 AD-71849 A-144253AAGAAUCCCAGUGGCUCCUdTdT 1178 A- AGGAGCCACUGGGAUUCUUdTdT 1492AAGAAUCCCAGUGGCUCCU 1806 144254 AD-71850 A-144255UGGGAAGACUGGGUCACAAdTdT 1179 A- UUGUGACCCAGUCUUCCCAdTdT 1493UGGGAAGACUGGGUCACAG 1807 144256 AD-71851 A-144257ACAGCUGCCUACAUGGACAdTdT 1180 A- UGUCCAUGUAGGCAGCUGUdTdT 1494ACAGCUGCCUACAUGGACA 1808 144258 AD-71852 A-144259ACAGUGAGCUUGUCUGCCAdTdT 1181 A- UGGCAGACAAGCUCACUGUdTdT 1495ACAGUGAGCUUGUCUGCCA 1809 144260 AD-71853 A-144261ACUGGGUUUUAUAGAACAAdTdT 1182 A- UUGUUCUAUAAAACCCAGUdTdT 1496ACUGGGUUUUAUAGAACAC 1810 144262 AD-71854 A-144263ACCCAAUCUGGGCUACAGAdTdT 1183 A- UCUGUAGCCCAGAUUGGGUdTdT 1497ACCCAAUCUGGGCUACAGC 1811 144264 AD-71855 A-144265CAGCUUUGAGACUAACUCAdTdT 1184 A- UGAGUUAGUCUCAAAGCUGdTdT 1498CAGCUUUGAGACUAACUCA 1812 144266 AD-71856 A-144267GGGAACCCCUUCCACUACUdTdT 1185 A- AGUAGUGGAAGGGGUUCCCdTdT 1499GGGAACCCCUUCCACUACU 1813 144268 AD-71857 A-144269CUUCAGCUAUGGGGUGGCUdTdT 1186 A- AGCCACCCCAUAGCUGAAGdTdT 1500CUUCAGCUAUGGGGUGGCU 1814 144270 AD-71858 A-144271CUUGCUCUGAAGUAGAAAUdTdT 1187 A- AUUUCUACUUCAGAGCAAGdTdT 1501CUUGCUCUGAAGUAGAAAU 1815 144272 AD-71859 A-144273AAAUCGACUGCCUAACAGAdTdT 1188 A- UCUGUUAGGCAGUCGAUUUdTdT 1502AAAUCGACUGCCUAACAGG 1816 144274 AD-71860 A-144275GAGAUCAUAAGAACCUCCAdTdT 1189 A- UGGAGGUUCUUAUGAUCUCdTdT 1503GAGAUCAUAAGAACCUCCG 1817 144276 AD-71861 A-144277GCACAGAUAUUGUCAUGGAdTdT 1190 A- UCCAUGACAAUAUCUGUGCdTdT 1504GCACAGAUAUUGUCAUGGA 1818 144278 AD-71862 A-144279UGGAUGUUGGCUCCAGUCUdTdT 1191 A- AGACUGGAGCCAACAUCCAdTdT 1505UGGAUGUUGGCUCCAGUCU 1819 144280 AD-71863 A-144281UAAACCCUGCCAUUGAUAUdTdT 1192 A- AUAUCAAUGGCAGGGUUUAdTdT 1506UAAACCCUGCCAUUGAUAU 1820 144282 AD-71864 A-144283UUGGACAGGUGGAAGGGGAdTdT 1193 A- UCCCCUUCCACCUGUCCAAdTdT 1507UUGGACAGGUGGAAGGGGC 1821 144284 AD-71865 A-144285GGGCAUUUGUCCAGGGCCUdTdT 1194 A- AGGCCCUGGACAAAUGCCCdTdT 1508GGGCAUUUGUCCAGGGCCU 1822 144286 AD-71866 A-144287UUGGCCUCUUCACCCUAGAdTdT 1195 A- UCUAGGGUGAAGAGGCCAAdTdT 1509UUGGCCUCUUCACCCUAGA 1823 144288 AD-71867 A-144289AGAGGAGCUACACUAUUCAdTdT 1196 A- UGAAUAGUGUAGCUCCUCUdTdT 1510AGAGGAGCUACACUAUUCC 1824 144290 AD-71868 A-144291CCCCGAGGGGAGCCUGCAAdTdT 1197 A- UUGCAGGCUCCCCUCGGGGdTdT 1511CCCCGAGGGGAGCCUGCAC 1825 144292 AD-71869 A-144293CACACCCGUGGCCCUAGCAdTdT 1198 A- UGCUAGGGCCACGGGUGUGdTdT 1512CACACCCGUGGCCCUAGCA 1826 144294 AD-71870 A-144295ACCUACAAGAUCCCGGCAUdTdT 1199 A- AUGCCGGGAUCUUGUAGGUdTdT 1513ACCUACAAGAUCCCGGCAU 1827 144296 AD-71871 A-144297UUUGGCAGCAUCCCCAUUAdTdT 1200 A- UAAUGGGGAUGCUGCCAAAdTdT 1514UUUGGCAGCAUCCCCAUUG 1828 144298 AD-71872 A-144301CCUGCUCCGCGACUGCCCAdTdT 1201 A- UGGGCAGUCGCGGAGCAGGdTdT 1515CCUGCUCCGCGACUGCCCC 1829 144302 AD-71873 A-144303CAACAAGAAGGCCAUCUAUdTdT 1202 A- AUAGAUGGCCUUCUUGUUGdTdT 1516CAACAAGAAGGCCAUCUAU 1830 144304 AD-71874 A-144305UAUGCAUCGAAGGCUGUUAdTdT 1203 A- UAACAGCCUUCGAUGCAUAdTdT 1517UAUGCAUCGAAGGCUGUUG 1831 144306 AD-71875 A-144307UGGAGAGCCGCCCCUCUUAdTdT 1204 A- UAAGAGGGGCGGCUCUCCAdTdT 1518UGGAGAGCCGCCCCUCUUC 1832 144308 AD-71876 A-144309UCCUGGCUGCUUCUAUCUUdTdT 1205 A- AAGAUAGAAGCAGCCAGGAdTdT 1519UCCUGGCUGCUUCUAUCUU 1833 144310 AD-71877 A-144311UUCUUUGCCAUCAAAGAUAdTdT 1206 A- UAUCUUUGAUGGCAAAGAAdTdT 1520UUCUUUGCCAUCAAAGAUG 1834 144312 AD-71878 A-144313CCAUCCGUGCAGCUCGAGAdTdT 1207 A- UCUCGAGCUGCACGGAUGGdTdT 1521CCAUCCGUGCAGCUCGAGC 1835 144314 AD-71879 A-144315AGCUCAGCACACAGGUAAUdTdT 1208 A- AUUACCUGUGUGCUGAGCUdTdT 1522AGCUCAGCACACAGGUAAU 1836 144316 AD-71880 A-144317AAUAACGUGAAGGAACUCUdTdT 1209 A- AGAGUUCCUUCACGUUAUUdTdT 1523AAUAACGUGAAGGAACUCU 1837 144318 AD-71881 A-144319UUCCGGCUAGACAGCCCUAdTdT 1210 A- UAGGGCUGUCUAGCCGGAAdTdT 1524UUCCGGCUAGACAGCCCUG 1838 144320 AD-71882 A-144321UGCCACCCCGGAGAAGAUAdTdT 1211 A- UAUCUUCUCCGGGGUGGCAdTdT 1525UGCCACCCCGGAGAAGAUC 1839 144322 AD-71883 A-144323AUCCGCAAUGCCUGCGUGAdTdT 1212 A- UCACGCAGGCAUUGCGGAUdTdT 1526AUCCGCAAUGCCUGCGUGG 1840 144324 AD-71884 A-144325GACAAGUUCACCACCCUGUdTdT 1213 A- ACAGGGUGGUGAACUUGUCdTdT 1527GACAAGUUCACCACCCUGU 1841 144326 AD-71885 A-144327UGUGUGUCACUGGUGUCCAdTdT 1214 A- UGGACACCAGUGACACACAdTdT 1528UGUGUGUCACUGGUGUCCC 1842 144328 AD-71886 A-144329AGAAAACUGCAAACCCUGAdTdT 1215 A- UCAGGGUUUGCAGUUUUCUdTdT 1529AGAAAACUGCAAACCCUGG 1843 144330 AD-71887 A-144331CUGGUCUGUGAGGGUCUAAdTdT 1216 A- UUAGACCCUCACAGACCAGdTdT 1530CUGGUCUGUGAGGGUCUAA 1844 144332 AD-71888 A-144333AAAGAGAGAGUCCUCAGCAdTdT 1217 A- UGCUGAGGACUCUCUCUUUdTdT 1531AAAGAGAGAGUCCUCAGCA 1845 144334 AD-71889 A-144335AGAGUCUUCUUGUGCUGCAdTdT 1218 A- UGCAGCACAAGAAGACUCUdTdT 1532AGAGUCUUCUUGUGCUGCC 1846 144336 AD-71890 A-144337GCCUUUGGGCUUCCAUGGAdTdT 1219 A- UCCAUGGAAGCCCAAAGGCdTdT 1533GCCUUUGGGCUUCCAUGGA 1847 144338 AD-71891 A-144341CAGAACAUGGAUCUAUUAAdTdT 1220 A- UUAAUAGAUCCAUGUUCUGdTdT 1534CAGAACAUGGAUCUAUUAA 1848 144342 AD-71892 A-144343UUAAAGUCACAGAAUGACAdTdT 1221 A- UGUCAUUCUGUGACUUUAAdTdT 1535UUAAAGUCACAGAAUGACA 1849 144344 AD-71893 A-144345AGACCUGUGAUUUGUCAAAdTdT 1222 A- UUUGACAAAUCACAGGUCUdTdT 1536AGACCUGUGAUUUGUCAAG 1850 144346 AD-71894 A-144347AGAUGGGAUUUGGAAGACAdTdT 1223 A- UGUCUUCCAAAUCCCAUCUdTdT 1537AGAUGGGAUUUGGAAGACA 1851 144348 AD-71895 A-144349AAGUGAAUGCAAUGGAAGAdTdT 1224 A- UCUUCCAUUGCAUUCACUUdTdT 1538AAGUGAAUGCAAUGGAAGA 1852 144350 AD-71896 A-144351AAGAUUUUGAUCAAAAAUAdTdT 1225 A- UAUUUUUGAUCAAAAUCUUdTdT 1539AAGAUUUUGAUCAAAAAUG 1853 144352 AD-71897 A-144353UGUAAUUUGUAAACACAAUdTdT 1226 A- AUUGUGUUUACAAAUUACAdTdT 1540UGUAAUUUGUAAACACAAU 1854 144354 AD-71898 A-144355GAUAAGCAAAUUCAAAACUdTdT 1227 A- AGUUUUGAAUUUGCUUAUCdTdT 1541GAUAAGCAAAUUCAAAACU 1855 144356 AD-71899 A-144357ACUGUUAUGCCUAAAUGGUdTdT 1228 A- ACCAUUUAGGCAUAACAGUdTdT 1542ACUGUUAUGCCUAAAUGGU 1856 144358 AD-71900 A-144359UGAAUAUGCAAUUAGGAUAdTdT 1229 A- UAUCCUAAUUGCAUAUUCAdTdT 1543UGAAUAUGCAAUUAGGAUC 1857 144360 AD-71901 A-144361AUCAUUUUCUGUCUGUUUUdTdT 1230 A- AAAACAGACAGAAAAUGAUdTdT 1544AUCAUUUUCUGUCUGUUUU 1858 144362 AD-71902 A-144363UUAAUCAUGUAUCUGGAAUdTdT 1231 A- AUUCCAGAUACAUGAUUAAdTdT 1545UUAAUCAUGUAUCUGGAAU 1859 144364 AD-71903 A-144365AAUAGGGUCGGGAAGGGUUdTdT 1232 A- AACCCUUCCCGACCCUAUUdTdT 1546AAUAGGGUCGGGAAGGGUU 1860 144366 AD-71904 A-144367UUUGUGCUAUUCCCCACUUdTdT 1233 A- AAGUGGGGAAUAGCACAAAdTdT 1547UUUGUGCUAUUCCCCACUU 1861 144368 AD-71905 A-144369UUACUGGACAGCCUGUAUAdTdT 1234 A- UAUACAGGCUGUCCAGUAAdTdT 1548UUACUGGACAGCCUGUAUA 1862 144370 AD-71906 A-144371AACCUCAAGUUCUGAUGGUdTdT 1235 A- ACCAUCAGAACUUGAGGUUdTdT 1549AACCUCAAGUUCUGAUGGU 1863 144372 AD-71907 A-144373UGUCUGUCCUUUGAAGAGAdTdT 1236 A- UCUCUUCAAAGGACAGACAdTdT 1550UGUCUGUCCUUUGAAGAGG 1864 144374 AD-71908 A-144375AGGAUUCCCACAAACCUCUdTdT 1237 A- AGAGGUUUGUGGGAAUCCUdTdT 1551AGGAUUCCCACAAACCUCU 1865 144376 AD-71909 A-144377UAGAAGCUUAAACCGAAGUdTdT 1238 A- ACUUCGGUUUAAGCUUCUAdTdT 1552UAGAAGCUUAAACCGAAGU 1866 144378 AD-71910 A-144379AAGUUACUUUAAAUCGUGUdTdT 1239 A- ACACGAUUUAAAGUAACUUdTdT 1553AAGUUACUUUAAAUCGUGU 1867 144380 AD-71911 A-144381UGCCUUCCUGUGAAAGCCUdTdT 1240 A- AGGCUUUCACAGGAAGGCAdTdT 1554UGCCUUCCUGUGAAAGCCU 1868 144382 AD-71912 A-144383CUGGCCUUCAAACCAAUGAdTdT 1241 A- UCAUUGGUUUGAAGGCCAGdTdT 1555CUGGCCUUCAAACCAAUGA 1869 144384 AD-71913 A-144385AACAGCAAAGCAUAACCUUdTdT 1242 A- AAGGUUAUGCUUUGCUGUUdTdT 1556AACAGCAAAGCAUAACCUU 1870 144386 AD-71914 A-144387UUGAAUCUAUACUCAAAUUdTdT 1243 A- AAUUUGAGUAUAGAUUCAAdTdT 1557UUGAAUCUAUACUCAAAUU 1871 144388 AD-71915 A-144389UUUUGCAAUGAGGCAGUGAdTdT 1244 A- UCACUGCCUCAUUGCAAAAdTdT 1558UUUUGCAAUGAGGCAGUGG 1872 144390 AD-71916 A-144391UGGGGUAAGGUUAAAUCCUdTdT 1245 A- AGGAUUUAACCUUACCCCAdTdT 1559UGGGGUAAGGUUAAAUCCU 1873 144392 AD-71917 A-144393UCUAACCAUCUUUGAAUCAdTdT 1246 A- UGAUUCAAAGAUGGUUAGAdTdT 1560UCUAACCAUCUUUGAAUCA 1874 144394 AD-71918 A-144395AUCAUUGGAAAGAAUAAAGdTdT 1247 A- CUUUAUUCUUUCCAAUGAUdTdT 1561AUCAUUGGAAAGAAUAAAG 1875 144396 AD-71919 A-144397AAUGAAACAAAUUCAAGGUdTdT 1248 A- ACCUUGAAUUUGUUUCAUUdTdT 1562AAUGAAACAAAUUCAAGGU 1876 144398 AD-71920 A-144399AGGUUAAUUGGAUCUGAUUdTdT 1249 A- AAUCAGAUCCAAUUAACCUdTdT 1563AGGUUAAUUGGAUCUGAUU 1877 144400 AD-71921 A-144401UUUGUGAAGCUGCAUAAAGdTdT 1250 A- CUUUAUGCAGCUUCACAAAdTdT 1564UUUGUGAAGCUGCAUAAAG 1878 144402 AD-71922 A-144403AGCAAGAUUACUCUAUAAUdTdT 1251 A- AUUAUAGAGUAAUCUUGCUdTdT 1565AGCAAGAUUACUCUAUAAU 1879 144404 AD-71923 A-144405UACAAAAAUCCAACCAACUdTdT 1252 A- AGUUGGUUGGAUUUUUGUAdTdT 1566UACAAAAAUCCAACCAACU 1880 144406 AD-71924 A-144407ACUCAAUUAUUGAGCACGUdTdT 1253 A- ACGUGCUCAAUAAUUGAGUdTdT 1567ACUCAAUUAUUGAGCACGU 1881 144408 AD-71925 A-144409UACAAUGUUCUAGAUUUCUdTdT 1254 A- AGAAAUCUAGAACAUUGUAdTdT 1568UACAAUGUUCUAGAUUUCU 1882 144410 AD-71926 A-144411UUCUUUCCCUUCCUCUUUAdTdT 1255 A- UAAAGAGGAAGGGAAAGAAdTdT 1569UUCUUUCCCUUCCUCUUUG 1883 144412 AD-71927 A-144413GAAGAGAAUAUUUGUAUUAdTdT 1256 A- UAAUACAAAUAUUCUCUUCdTdT 1570GAAGAGAAUAUUUGUAUUC 1884 144414 AD-71928 A-144415UUCCAAAUACUCUUUGAGUdTdT 1257 A- ACUCAAAGAGUAUUUGGAAdTdT 1571UUCCAAAUACUCUUUGAGU 1885 144416 AD-71929 A-144417UAUUUACAAAAAAGAUUAUdTdT 1258 A- AUAAUCUUUUUUGUAAAUAdTdT 1572UAUUUACAAAAAAGAUUAU 1886 144418 AD-72020 A-144419UAUGUUUAAUCUUUACAUUdTdT 1259 A- AAUGUAAAGAUUAAACAUAdTdT 1573UAUGUUUAAUCUUUACAUU 1887 144420 AD-72021 A-144421UUUGAAGCCAAAGUAAUUUdTdT 1260 A- AAAUUACUUUGGCUUCAAAdTdT 1574UUUGAAGCCAAAGUAAUUU 1888 144422 AD-72022 A-144423UUCCACCUAGAAAUGAUGAdTdT 1261 A- UCAUCAUUUCUAGGUGGAAdTdT 1575UUCCACCUAGAAAUGAUGC 1889 144424 AD-72023 A-144425UAUCAGUCCUGGCAUGGUAdTdT 1262 A- UACCAUGCCAGGACUGAUAdTdT 1576UAUCAGUCCUGGCAUGGUG 1890 144426 AD-72024 A-144427UGGCUCACCCCUAUAAUCAdTdT 1263 A- UGAUUAUAGGGGUGAGCCAdTdT 1577UGGCUCACCCCUAUAAUCC 1891 144428 AD-72025 A-144429AUCCCAGCACUUUGGGAGAdTdT 1264 A- UCUCCCAAAGUGCUGGGAUdTdT 1578AUCCCAGCACUUUGGGAGG 1892 144430 AD-72026 A-144431CUAAGGCAGGAGAAUUGCUdTdT 1265 A- AGCAAUUCUCCUGCCUUAGdTdT 1579CUAAGGCAGGAGAAUUGCU 1893 144432 AD-72027 A-144433UGCUUGAGCCCAGCAGUUUdTdT 1266 A- AAACUGCUGGGCUCAAGCAdTdT 1580UGCUUGAGCCCAGCAGUUU 1894 144434 AD-72028 A-144435UUGAGACCAGCCUGGGCAAdTdT 1267 A- UUGCCCAGGCUGGUCUCAAdTdT 1581UUGAGACCAGCCUGGGCAA 1895 144436 AD-72029 A-144437ACAUAGAGAGCUCCUGUCUdTdT 1268 A- AGACAGGAGCUCUCUAUGUdTdT 1582ACAUAGAGAGCUCCUGUCU 1896 144438 AD-72030 A-144439UCUUUAAAAAAAAUUUUUUdTdT 1269 A- AAAAAAUUUUUUUUAAAGAdTdT 1583UCUUUAAAAAAAAUUUUUU 1897 144440 AD-72031 A-144441UUAAUUAGUUGGUCUUGAUdTdT 1270 A- AUCAAGACCAACUAAUUAAdTdT 1584UUAAUUAGUUGGUCUUGAU 1898 144442 AD-72032 A-144443UAGUGCAUGCCUGUAGUCAdTdT 1271 A- UGACUACAGGCAUGCACUAdTdT 1585UAGUGCAUGCCUGUAGUCC 1899 144444 AD-72033 A-144445CCCAACUACUUGAAAGGCUdTdT 1272 A- AGCCUUUCAAGUAGUUGGGdTdT 1586CCCAACUACUUGAAAGGCU 1900 144446 AD-72034 A-144447CUGAGGUGGAGAGAUCAUUdTdT 1273 A- AAUGAUCUCUCCACCUCAGdTdT 1587CUGAGGUGGAGAGAUCAUU 1901 144448 AD-72035 A-144449UUUGAGCUCAGGAGGUUGAdTdT 1274 A- UCAACCUCCUGAGCUCAAAdTdT 1588UUUGAGCUCAGGAGGUUGA 1902 144450 AD-72036 A-144451UUGAGGCUGCAGUGAGCUAdTdT 1275 A- UAGCUCACUGCAGCCUCAAdTdT 1589UUGAGGCUGCAGUGAGCUA 1903 144452 AD-72037 A-144455CUCCUGCCUGAGCGACUGAdTdT 1276 A- UCAGUCGCUCAGGCAGGAGdTdT 1590CUCCUGCCUGAGCGACUGA 1904 144456 AD-72038 A-144457UGAGCAAGAUCUUGUCUCUdTdT 1277 A- AGAGACAAGAUCUUGCUCAdTdT 1591UGAGCAAGAUCUUGUCUCU 1905 144458 AD-72039 A-144459UGAAGAAAAAAAAAGAAAUdTdT 1278 A- AUUUCUUUUUUUUUCUUCAdTdT 1592UGAAGAAAAAAAAAGAAAU 1906 144460 AD-72040 A-144461UAAAAAUGCUGCUAUCAAAdTdT 1279 A- UUUGAUAGCAGCAUUUUUAdTdT 1593UAAAAAUGCUGCUAUCAAA 1907 144462 AD-72041 A-144463AAAAUCAAGCCCAACCAGAdTdT 1280 A- UCUGGUUGGGCUUGAUUUUdTdT 1594AAAAUCAAGCCCAACCAGA 1908 144464 AD-72042 A-144465AGAGGUAGAAGAGCCAAGAdTdT 1281 A- UCUUGGCUCUUCUACCUCUdTdT 1595AGAGGUAGAAGAGCCAAGA 1909 144466 AD-72043 A-144467AAGCCUGGGUUCUCAUCCUdTdT 1282 A- AGGAUGAGAACCCAGGCUUdTdT 1596AAGCCUGGGUUCUCAUCCU 1910 144468 AD-72044 A-144469CUAGCUCUGUCUCUUCUGUdTdT 1283 A- ACAGAAGAGACAGAGCUAGdTdT 1597CUAGCUCUGUCUCUUCUGU 1911 144470 AD-72045 A-144473UUGGACUGUCAAUUCCCCUdTdT 1284 A- AGGGGAAUUGACAGUCCAAdTdT 1598UUGGACUGUCAAUUCCCCU 1912 144474 AD-72046 A-144475CCUUCCUGUGAUCCAUUUUdTdT 1285 A- AAAAUGGAUCACAGGAAGGdTdT 1599CCUUCCUGUGAUCCAUUUU 1913 144476 AD-72047 A-144481UCUCACGUCUUCUGCUUUAdTdT 1286 A- UAAAGCAGAAGACGUGAGAdTdT 1600UCUCACGUCUUCUGCUUUA 1914 144482 AD-72048 A-144485UAUGACCUGAAAACUCCAAdTdT 1287 A- UUGGAGUUUUCAGGUCAUAdTdT 1601UAUGACCUGAAAACUCCAG 1915 144486 AD-72049 A-144487UUACAUAAAGGAUCUGCAAdTdT 1288 A- UUGCAGAUCCUUUAUGUAAdTdT 1602UUACAUAAAGGAUCUGCAG 1916 144488 AD-72050 A-144489CAGCUAUCUAAGGCUUGGUdTdT 1289 A- ACCAAGCCUUAGAUAGCUGdTdT 1603CAGCUAUCUAAGGCUUGGU 1917 144490 AD-72051 A-144493AUGAUACCUGGGUCUAAUAdTdT 1290 A- UAUUAGACCCAGGUAUCAUdTdT 1604AUGAUACCUGGGUCUAAUG 1918 144494 AD-72052 A-144495AACUCUGCUGAGAUCACCUdTdT 1291 A- AGGUGAUCUCAGCAGAGUUdTdT 1605AACUCUGCUGAGAUCACCU 1919 144496 AD-72053 A-144497ACCUCAAGUUUCUGCGGUUdTdT 1292 A- AACCGCAGAAACUUGAGGUdTdT 1606ACCUCAAGUUUCUGCGGUU 1920 144498 AD-72054 A-144499UUGGUAAAGAGAACAAGGAdTdT 1293 A- UCCUUGUUCUCUUUACCAAdTdT 1607UUGGUAAAGAGAACAAGGG 1921 144500 AD-72055 A-144501AAGAACAAACAUCCCUUUUdTdT 1294 A- AAAAGGGAUGUUUGUUCUUdTdT 1608AAGAACAAACAUCCCUUUU 1922 144502 AD-72056 A-144503UUUUAUUGCUCCAAAUGGUdTdT 1295 A- ACCAUUUGGAGCAAUAAAAdTdT 1609UUUUAUUGCUCCAAAUGGU 1923 144504 AD-72057 A-144505GAUUUAAUCCCUACAUGGUdTdT 1296 A- ACCAUGUAGGGAUUAAAUCdTdT 1610GAUUUAAUCCCUACAUGGU 1924 144506 AD-72058 A-144507GGUGCUGGGUGGACAAUGUdTdT 1297 A- ACAUUGUCCACCCAGCACCdTdT 1611GGUGCUGGGUGGACAAUGU 1925 144508 AD-72059 A-144509UGUCACUGUCACAUGCCUUdTdT 1298 A- AAGGCAUGUGACAGUGACAdTdT 1612UGUCACUGUCACAUGCCUU 1926 144510 AD-72060 A-144511UUCACUGUAUAAAUCCAAAdTdT 1299 A- UUUGGAUUUAUACAGUGAAdTdT 1613UUCACUGUAUAAAUCCAAC 1927 144512 AD-72061 A-144513ACCUUCUGCCAGAGAGAAUdTdT 1300 A- AUUCUCUCUGGCAGAAGGUdTdT 1614ACCUUCUGCCAGAGAGAAU 1928 144514 AD-72062 A-144517CAUGGAGGGAGGAUAGUGAdTdT 1301 A- UCACUAUCCUCCCUCCAUGdTdT 1615CAUGGAGGGAGGAUAGUGG 1929 144518 AD-72063 A-144519AAAUGAUAUAGUUGGACUAdTdT 1302 A- UAGUCCAACUAUAUCAUUUdTdT 1616AAAUGAUAUAGUUGGACUG 1930 144520 AD-72064 A-144521ACUGGUGCUUGAUGUCACUdTdT 1303 A- AGUGACAUCAAGCACCAGUdTdT 1617ACUGGUGCUUGAUGUCACU 1931 144522 AD-72065 A-144523CUAAUAAAUGAAACUGUCAdTdT 1304 A- UGACAGUUUCAUUUAUUAGdTdT 1618CUAAUAAAUGAAACUGUCA 1932 144524

TABLE 8 XDH Single Dose (10 nM) Screen in Primary Human HepatocytesDuplex Position in Name AVG STDEV NM_000379.3 AD-71930 82.8 4.5  18-36AD-71931 56.8 3.9  36-54 AD-71932 60.4 4.8  69-87 AD-71933 49.5 5.4 86-104 AD-71934 65.8 15.1  104-122 AD-71935 50.8 6.2  119-137 AD-7193665.3 19.6  154-172 AD-71937 94.4 19.3  172-190 AD-71938 90.8 1.8 188-206 AD-71939 80.7 9.1  205-223 AD-71940 67.2 2.3  223-241 AD-7194170.8 23.2  239-257 AD-71942 49.6 8.9  273-291 AD-71943 50.3 3.7  291-309AD-71944 52.9 6.5  308-326 AD-71945 79.4 5.0  325-343 AD-71946 92.7 0.1 342-360 AD-71947 50.1 17.5  358-376 AD-71948 65.1 19.8  376-394AD-71949 87.7 0.1  392-410 AD-71950 44.1 2.8  410-428 AD-71951 70.6 5.2 425-443 AD-71952 43.5 4.0  444-462 AD-71953 85.3 1.7  459-477 AD-7195456.1 7.2  477-495 AD-71955 80.7 22.7  495-513 AD-71956 81.6 9.6  510-528AD-71957 69.1 8.1  529-547 AD-71958 60.6 2.7  544-562 AD-71959 79.8 12.1 563-581 AD-71960 68.4 16.0  580-598 AD-71961 51.1 2.0  596-614 AD-7196252.0 0.7  630-648 AD-71963 56.9 3.6  647-665 AD-71964 52.4 3.4  663-681AD-71965 90.2 29.6  680-698 AD-71966 53.2 5.5  697-715 AD-71967 54.5 4.2 714-732 AD-71968 53.3 3.2  731-749 AD-71969 57.0 4.7  748-766 AD-7197072.1 10.5  765-783 AD-71971 60.0 4.2  783-801 AD-71972 86.7 6.8  799-817AD-71973 89.1 7.0  816-834 AD-71974 65.9 6.2  834-852 AD-71975 62.3 16.3 852-870 AD-71976 75.7 22.3  869-887 AD-71977 69.5 17.5  884-902AD-71978 112.9 17.2  902-920 AD-71979 88.8 12.2  919-937 AD-71980 47.220.4  936-954 AD-71981 51.9 9.6  953-971 AD-71982 46.5 1.2  969-987AD-71983 58.2 14.4  986-1004 AD-71984 61.3 5.4 1005-1023 AD-71985 78.27.6 1021-1039 AD-71986 84.0 15.2 1037-1055 AD-71987 66.6 4.0 1056-1074AD-71988 56.9 3.6 1071-1089 AD-71989 61.2 31.8 1088-1106 AD-71990 33.54.7 1105-1123 AD-71991 77.8 4.1 1123-1141 AD-71992 76.4 2.3 1141-1159AD-71993 34.5 16.4 1158-1176 AD-71994 83.5 18.6 1173-1191 AD-71995 53.32.1 1190-1208 AD-71996 53.9 2.9 1207-1225 AD-71997 83.6 1.6 1225-1243AD-71998 39.5 0.4 1242-1260 AD-71999 58.1 7.4 1259-1277 AD-72000 87.322.1 1277-1295 AD-72001 89.2 9.7 1292-1310 AD-72002 84.3 6.7 1310-1328AD-72003 40.5 6.9 1326-1344 AD-72004 55.2 17.3 1343-1361 AD-72005 66.620.9 1362-1380 AD-72006 42.7 0.0 1378-1396 AD-72007 32.6 6.0 1394-1412AD-72008 76.2 10.4 1411-1429 AD-72009 57.3 2.5 1428-1446 AD-72010 86.36.4 1445-1463 AD-72011 56.5 13.4 1463-1481 AD-72012 48.8 5.3 1481-1499AD-72013 52.2 1.6 1496-1514 AD-72014 45.8 3.6 1515-1533 AD-72015 46.15.6 1530-1548 AD-72016 78.2 7.7 1548-1566 AD-72017 100.3 8.8 1564-1582AD-72018 68.4 4.1 1583-1601 AD-72019 90.5 1.4 1598-1616 AD-71752 60.914.0 1617-1635 AD-71753 63.1 2.1 1634-1652 AD-71754 108.1 31.4 1667-1685AD-71755 84.4 21.3 1685-1703 AD-71756 59.5 6.7 1702-1720 AD-71757 48.50.7 1718-1736 AD-71758 92.8 6.3 1734-1752 AD-71759 73.0 7.8 1752-1770AD-71760 63.9 14.0 1770-1788 AD-71761 108.8 2.1 1787-1805 AD-71762 124.621.3 1803-1821 AD-71763 55.1 0.0 1819-1837 AD-71764 121.9 19.7 1836-1854AD-71765 41.8 7.0 1854-1872 AD-71766 44.0 2.0 1872-1890 AD-71767 51.30.7 1889-1907 AD-71768 58.3 3.5 1921-1939 AD-71769 53.5 9.7 1939-1957AD-71770 74.2 6.2 1973-1991 AD-71771 60.7 13.6 1991-2009 AD-71772 58.411.4 2040-2058 AD-71773 77.4 12.1 2057-2075 AD-71774 130.8 7.0 2075-2093AD-71775 75.8 7.8 2091-2109 AD-71776 76.4 3.0 2110-2128 AD-71777 76.027.4 2126-2144 AD-71778 48.3 9.2 2142-2160 AD-71779 43.1 10.8 2177-2195AD-71780 78.4 16.8 2194-2212 AD-71781 71.2 14.3 2210-2228 AD-71782 92.83.7 2228-2246 AD-71783 60.8 19.1 2244-2262 AD-71784 62.8 17.1 2263-2281AD-71785 58.7 13.7 2280-2298 AD-71786 50.7 5.7 2295-2313 AD-71787 46.75.2 2314-2332 AD-71788 60.8 0.6 2331-2349 AD-71789 105.1 1.0 2347-2365AD-71790 55.1 18.8 2364-2382 AD-71791 72.4 10.2 2381-2399 AD-71792 72.910.4 2399-2417 AD-71793 61.5 5.2 2414-2432 AD-71794 65.0 6.0 2431-2449AD-71795 69.6 23.1 2448-2466 AD-71796 68.0 9.0 2467-2485 AD-71797 67.818.4 2483-2501 AD-71798 91.7 21.0 2500-2518 AD-71799 57.4 9.0 2517-2535AD-71800 64.3 13.5 2550-2568 AD-71801 40.3 9.0 2567-2585 AD-71802 80.35.6 2586-2604 AD-71803 51.1 6.2 2602-2620 AD-71804 90.3 6.2 2618-2636AD-71805 51.4 5.3 2637-2655 AD-71806 86.3 18.5 2654-2672 AD-71807 68.87.7 2670-2688 AD-71808 69.9 45.6 2686-2704 AD-71809 51.2 7.3 2705-2723AD-71810 36.8 0.6 2722-2740 AD-71811 52.1 4.3 2739-2757 AD-71812 76.99.4 2754-2772 AD-71813 55.9 2.2 2773-2791 AD-71814 68.7 14.7 2788-2806AD-71815 90.8 18.1 2805-2823 AD-71816 71.1 9.0 2823-2841 AD-71817 87.49.4 2840-2858 AD-71818 67.1 15.4 2858-2876 AD-71819 79.7 16.7 2875-2893AD-71820 38.2 0.7 2891-2909 AD-71821 58.9 18.2 2909-2927 AD-71822 55.718.0 2924-2942 AD-71823 45.3 3.3 2941-2959 AD-71824 57.1 6.9 2958-2976AD-71825 68.3 7.7 2977-2995 AD-71826 47.3 11.1 2993-3011 AD-71827 51.66.0 3011-3029 AD-71828 51.5 1.5 3026-3044 AD-71829 85.5 6.7 3044-3062AD-71830 43.1 1.9 3062-3080 AD-71831 36.7 2.9 3079-3097 AD-71832 77.04.6 3095-3113 AD-71833 44.9 4.0 3112-3130 AD-71834 41.7 2.7 3129-3147AD-71835 53.3 1.6 3146-3164 AD-71836 52.5 1.0 3164-3182 AD-71837 39.10.2 3197-3215 AD-71838 93.7 2.2 3215-3233 AD-71839 55.6 2.8 3230-3248AD-71840 42.4 15.8 3247-3265 AD-71841 69.4 24.3 3266-3284 AD-71842 69.61.7 3281-3299 AD-71843 51.5 1.0 3298-3316 AD-71844 43.0 0.9 3316-3334AD-71845 58.5 0.8 3333-3351 AD-71846 73.4 7.1 3350-3368 AD-71847 45.40.0 3366-3384 AD-71848 65.6 13.7 3384-3402 AD-71849 105.6 19.0 3401-3419AD-71850 69.2 12.5 3419-3437 AD-71851 54.0 2.2 3434-3452 AD-71852 51.16.3 3452-3470 AD-71853 71.3 1.4 3470-3488 AD-71854 48.8 0.3 3487-3505AD-71855 48.2 13.7 3502-3520 AD-71856 59.5 7.3 3521-3539 AD-71857 54.26.4 3538-3556 AD-71858 51.4 12.0 3555-3573 AD-71859 52.3 8.5 3570-3588AD-71860 62.1 13.0 3588-3606 AD-71861 39.3 2.7 3606-3624 AD-71862 67.92.7 3621-3639 AD-71863 56.4 3.9 3639-3657 AD-71864 78.6 4.6 3657-3675AD-71865 49.1 4.8 3672-3690 AD-71866 59.1 2.3 3690-3708 AD-71867 50.95.4 3706-3724 AD-71868 60.3 6.2 3724-3742 AD-71869 76.3 12.0 3740-3758AD-71870 68.1 6.6 3758-3776 AD-71871 108.2 8.5 3776-3794 AD-71872 75.421.9 3808-3826 AD-71873 59.2 8.4 3826-3844 AD-71874 69.0 10.4 3842-3860AD-71875 68.1 11.6 3859-3877 AD-71876 60.1 11.7 3876-3894 AD-71877 89.93.1 3893-3911 AD-71878 49.8 11.3 3912-3930 AD-71879 73.0 1.7 3928-3946AD-71880 91.3 7.5 3944-3962 AD-71881 125.0 7.3 3962-3980 AD-71882 107.59.5 3979-3997 AD-71883 67.6 7.3 3995-4013 AD-71884 55.4 7.6 4013-4031AD-71885 66.6 5.9 4029-4047 AD-71886 79.7 3.1 4048-4066 AD-71887 47.09.8 4063-4081 AD-71888 102.9 3.1 4080-4098 AD-71889 51.2 10.0 4098-4116AD-71890 48.9 1.2 4114-4132 AD-71891 55.7 6.0 4150-4168 AD-71892 69.511.2 4165-4183 AD-71893 67.3 9.9 4183-4201 AD-71894 46.7 4.8 4200-4218AD-71895 64.3 15.6 4218-4236 AD-71896 110.4 32.1 4233-4251 AD-71897 60.41.8 4250-4268 AD-71898 63.0 5.0 4269-4287 AD-71899 55.0 3.0 4285-4303AD-71900 45.6 2.0 4303-4321 AD-71901 49.7 0.9 4319-4337 AD-71902 85.926.2 4336-4354 AD-71903 54.4 17.8 4352-4370 AD-71904 71.3 2.1 4369-4387AD-71905 49.8 3.9 4386-4404 AD-71906 66.9 6.3 4404-4422 AD-71907 63.68.7 4422-4440 AD-71908 57.2 8.6 4438-4456 AD-71909 56.2 10.6 4456-4474AD-71910 52.5 6.5 4471-4489 AD-71911 51.1 15.5 4489-4507 AD-71912 64.723.6 4506-4524 AD-71913 50.1 8.8 4524-4542 AD-71914 49.9 5.8 4541-4559AD-71915 82.6 20.9 4558-4576 AD-71916 67.3 21.4 4574-4592 AD-71917 74.434.4 4592-4610 AD-71918 77.8 14.0 4607-4625 AD-71919 116.4 22.24626-4644 AD-71920 78.8 18.0 4641-4659 AD-71921 100.5 5.4 4659-4677AD-71922 61.7 3.7 4676-4694 AD-71923 72.1 4.6 4694-4712 AD-71924 57.05.9 4710-4728 AD-71925 74.1 2.6 4728-4746 AD-71926 56.9 0.2 4743-4761AD-71927 70.8 8.6 4761-4779 AD-71928 106.9 17.1 4777-4795 AD-71929 90.18.4 4795-4813 AD-72020 86.0 3.3 4811-4829 AD-72021 69.7 18.9 4828-4846AD-72022 82.9 12.2 4845-4863 AD-72023 99.1 4.4 4864-4882 AD-72024 81.910.9 4881-4899 AD-72025 101.1 1.4 4896-4914 AD-72026 106.2 3.6 4915-4933AD-72027 88.7 4.0 4930-4948 AD-72028 91.2 5.9 4947-4965 AD-72029 84.23.4 4965-4983 AD-72030 121.3 30.1 4981-4999 AD-72031 95.1 5.1 4999-5017AD-72032 82.7 0.4 5017-5035 AD-72033 83.0 3.2 5034-5052 AD-72034 64.812.6 5051-5069 AD-72035 94.7 10.7 5068-5086 AD-72036 92.1 2.7 5083-5101AD-72037 78.6 3.9 5118-5136 AD-72038 85.2 6.3 5134-5152 AD-72039 87.52.9 5152-5170 AD-72040 69.6 8.1 5170-5188 AD-72041 80.7 0.0 5186-5204AD-72042 83.3 28.8 5202-5220 AD-72043 107.3 0.1 5220-5238 AD-72044 100.84.9 5237-5255 AD-72045 145.2 42.9 5272-5290 AD-72046 76.9 12.7 5288-5306AD-72047 62.9 1.3 5339-5357 AD-72048 66.6 5.3 5372-5390 AD-72049 74.62.5 5391-5409 AD-72050 96.8 32.6 5407-5425 AD-72051 76.7 4.2 5440-5458AD-72052 91.9 6.7 5459-5477 AD-72053 67.5 16.4 5474-5492 AD-72054 85.90.9 5491-5509 AD-72055 99.8 5.4 5510-5528 AD-72056 111.5 2.8 5525-5543AD-72057 98.9 9.6 5544-5562 AD-72058 82.8 19.6 5560-5578 AD-72059 89.533.9 5578-5596 AD-72060 91.8 0.5 5595-5613 AD-72061 104.8 18.8 5612-5630AD-72062 73.9 5.0 5644-5662 AD-72063 98.7 19.8 5663-5681 AD-72064 84.814.9 5678-5696 AD-72065 77.7 15.9 5695-5713

Example 4. In Vitro Determination IC₅₀ Values of XDH iRNA Agents inCynomolgus Monkey and Mouse Primary Hepatocytes

A series of siRNA-GalNAc3 conjugates targeting an XDH gene were analyzedfor target knockdown in cynomolgus monkey and mouse primary hepatocytes.The results are provided as IC₅₀ (nM) in Table 9.

TABLE 9 XDH siRNA IC₅₀ Values in Primary Cynomolgus Monkey and MouseHepatocytes Duplex Cyno Mouse AD-70042 0.0047 >10 nM AD-70016 0.0060.2345 AD-70030 0.0209 >10 nM AD-70044 0.0224 >10 nM AD-70050 0.0524 >10nM AD-70055 0.055 >10 nM AD-70033 0.0569 0.1231 AD-70026 0.0614 0.1901AD-70051 0.0859 >10 nM AD-70052 0.1409 >10 nM AD-70020 0.4017 4.4548AD-70018 0.5446 0.8168 AD-70023 0.6569 0.4775 AD-70024 >10 nM 0.5886

Example 5. In Vivo Effect of Single Dose Administration of XDH iRNAAgents in Mice

A series of siRNA-GalNAc3 conjugates targeting an XDH gene were designedand tested for the ability to knockdown expression of XDH mRNA in 6-8week old C57BL/6 female mice (n=3 per group). A single 1 mg/kg dose ofAD-70016, AD-70018, AD-70020, AD-70023, AD-70026, AD-70030, AD-70033; orPBS control, was administered subcutaneously to the mice on day 1. Onday 10, the mice were sacrificed to assess knockdown of XDH mRNA inliver by RT-PCR. The effect of a single dose of the indicated agents areprovided in Table 10. The data are presented as the XDH mRNA levelremaining relative to PBS.

TABLE 10 XDH siRNA Single Dose (1 mg/kg) mRNA Knockdown in Mouse LiverRelative siRNA mRNA St. Dev. PBS 100.0 4.43 AD-70016 22.73 6.03 AD-7001865.93 8.83 AD-70020 88.98 35.71 AD-70023 50.14 22.53 AD-70026 62.25 1.99AD-70030 84.46 10.67 AD-70033 36.57 9.98

Example 6. In Vivo Effect of Increasing Single Dose Administration ofXDH iRNA Agents in Mice

Male mice typically have higher serum levels of uric acid than femalemice. Therefore, the ability of dsRNA agents targeting XDH to knockdownXDH was also assessed in male mice. Of the duplexes tested, AD-70016 wasfound to be most effective in decreasing expression of XDH mRNA and wassubsequently analyzed in a dose-response study. C57BL/6 male mice, 6-8weeks of age (n=3-4 per group) were administered a single dose ofAD-70016 at 10, 3, 1, 0.3, or 0.1 mg/kg; or a PBS control. On day 10,the mice were sacrificed to assess knockdown of XDH mRNA in liver byRT-PCR. The effects of a single dose of AD-70016 at the concentrationsindicated are provided in Table 10. The data are presented as the XDHmRNA level remaining relative to PBS.

TABLE 11 XDH siRNA AD-70016 Single Dose mRNA Knockdown Dose Response inMouse Liver mg/kg Relative mRNA St. Dev. 0 (PBS) 100.0 11.28 10 14.891.53 3 15.54 3.95 1 29.71 5.32 0.3 52.12 7.01 0.1 73.13 7.64

Further studies demonstrated the durability of knockdown by AD-70016 forat least 6 weeks post a single 10 mg/kg dose in C57BL/6 male mice.

Finally, in a single dose study using higher doses of GalNAc3 conjugatedAD-70016 (3 mg/kg, 10 mg/kg, and 30 mg/kg), achieved robust knockdown ofmRNA at day 10 (15.35%, 9.42%, and 8.29% remaining, respectively).

Example 7. In Vivo Effect of Increasing Single Dose Administration ofXDH iRNA Agents in Rats

The AD-70016-GalNAc conjugate, which has a single nucleotide mismatch tothe rat XDH sequence, was analyzed in a dose-response study in rats.Male Sprague-Dawley rats, 6-8 weeks of age (n=4 per group) wereadministered a single dose of AD-70016 at 15, 5, 1.5, 0.5, or 0.15mg/kg; or a PBS control. On day 10, the rats were sacrificed to assessknockdown of XDH mRNA in liver by RT-PCR. The effects of a single doseof AD-70016 at the concentrations indicated are provided in Table 11.The data are presented as the XDH mRNA level remaining relative to PBS.

TABLE 12 XDH siRNA AD-70016 Single Dose mRNA Knockdown Dose Response inRat Liver mg/kg Relative mRNA St. Dev. 0 (PBS) 100 17.98 15 19.01 4.92 513.74 2.08 1.5 31.97 7.20 0.5 45.14 8.32 0.15 63.74 8.28

The results demonstrate that AD-70016 is effective in knocking down XDHexpression in rats.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

1. A double stranded ribonucleic acid (dsRNA) agent for inhibitingexpression of a xanthine dehydrogenase (XDH) gene, wherein said dsRNAagent comprises a sense strand and an antisense strand forming a doublestranded region at least 17 nucleotides in length, wherein said sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of nucleotides 490-512, 86-104, 273-291,339-358, 410-428, 444-462, 490-509, 493-512, 888-907, 936-954, 969-987,1105-1123, 1158-1176, 1242-1260, 1326-1344, 1357-1412, 1357-1379,1357-1376, 1360-1379, 1378-1412, 1378-1396, 1394-1412, 1481-1499,1515-1548, 1515-1533, 1530-1548, 1718-1736, 1783-1802, 1854-1890,1854-1872, 1872-1890, 2053-2072, 2077-2096, 2137-2160, 2137-2156,2142-2160, 2173-2206, 2173-2192, 2177-2195, 2184-2203, 2187-2206,2314-2332, 2567-2604, 2567-2585, 2585-2604, 2620-2640, 2620-2639,2621-2640, 2722-2740, 2891-2909, 2941-2975, 2941-2959, 2956-2975,2993-3011, 3025-3061, 3025-3044, 3042-3061, 3062-3110, 3062-3080,3079-3097, 3091-3110, 3112-3147, 3112-3130, 3129-3147, 3197-3215,3247-3265, 3316-3334, 3366-3384, 3487-3520, 3487-3505, 3502-3520,3606-3624, 3672-3690, 3891-3930, 3891-3910, 3893-3912, 3912-3930,4063-4081, 4114-4132, 4152-4171, 4200-4218, 4300-4337, 4300-4319,4303-4321, 4319-4337, 4386-4404, 4519-4538, 4541-4559, 4618-4637, or4703-4722 of the nucleotide sequence of SEQ ID NO:1 and said antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, whereinthe dsRNA agent comprises at least one nucleotide comprising anucleotide modification. 2.-4. (canceled)
 5. The dsRNA agent of claim 1,wherein the sense and antisense strands comprise nucleotide sequencesselected from the group consisting of any of the nucleotide sequenceslisted in any one of Tables 3, 4, 6, and
 7. 6. (canceled)
 7. The dsRNAagent of claim 1, wherein substantially all of the nucleotides of saidsense strand and substantially all of the nucleotides of said antisensestrand comprise a nucleotide modification.
 8. (canceled)
 9. (canceled)10. The dsRNA agent of claim 7, wherein all of the nucleotides of saidsense strand and all of the nucleotides of said antisense strandcomprise a nucleotide modification.
 11. The dsRNA agent of claim 10,wherein the modified nucleotides are independently selected from thegroup consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, and a nucleotidecomprising a 5′-phosphate mimic. 12.-15. (canceled)
 16. The dsRNA agentof claim 1, wherein each strand is no more than 30 nucleotides inlength.
 17. The dsRNA agent of claim 1, wherein at least one strandcomprises a 3′ overhang of at least 1 nucleotide: or at least 2nucleotides.
 18. (canceled)
 19. The dsRNA agent of claim 1, furthercomprising a ligand.
 20. The dsRNA agent of claim 19, wherein the ligandis conjugated to the 3′ end of the sense strand of the dsRNA agent. 21.The dsRNA agent of claim 19, wherein the ligand is anN-acetylgalactosamine (GalNAc) derivative.
 22. The dsRNA agent of claim21, wherein the ligand is


23. The dsRNA agent of claim 22, wherein the dsRNA agent is conjugatedto the ligand as shown in the following schematic

and, wherein X is O or S.
 24. The dsRNA agent of claim 23, wherein the Xis O. 25.-37. (canceled)
 38. The dsRNA agent of claim 1, wherein thedouble stranded region is 17-30 nucleotide pairs in length: 17-23nucleotide pairs in length: 17-25 nucleotide pairs in length: 23-27nucleotide pairs in length: 19-21 nucleotide pairs in length: or 21-23nucleotide pairs in length. 39-43. (canceled)
 44. The dsRNA agent ofclaim 1, wherein each strand has is 15-30 nucleotides in length: or19-30 nucleotides in length. 45.-51. (canceled)
 52. The dsRNA agent ofclaim 1, wherein said agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage. 53.-85.(canceled)
 86. A pharmaceutical composition for inhibiting expression ofan XDH gene comprising the dsRNA agent of claim
 1. 87.-92. (canceled)93. A method of inhibiting xanthine dehydrogenase (XDH) expression in acell, the method comprising contacting the cell with the dsRNA agent ofclaim 1, thereby inhibiting expression of the XDH gene in the cell.94.-96. (canceled)
 97. A method of treating a subject having a diseaseor disorder that would benefit from reduction in XDH expression, themethod comprising administering to the subject a therapeuticallyeffective amount of the dsRNA agent of claim 1, thereby treating saidsubject. 98.-109. (canceled)